WO2023194388A1 - Fusion proteins and their use against eimeria - Google Patents
Fusion proteins and their use against eimeria Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
Definitions
- Coccidiosis in poultry production is a US$10 billion problem. Via malnutrition, decreased weight-gain, and chicken mortality, the poultry industry is on average globally losing substantial revenue.
- the coccidiosis disease is caused by a genus of parasites known as Eimeria spp.
- the parasites infect new chicken hosts via the natural foraging habit of pecking the ground.
- the animals pick up parasite oocysts, which then sporulate in the intestines of the chicken.
- the parasite is described as a ‘sporozoite’.
- the sporozoite then move to the section of the gut where it may proliferate.
- the parasite invades a single chicken cell in the gut wall of the chicken intestine. Inside the chicken cell, the parasite multiplies before rupturing the cell. The increased number of parasites now invade other cells of the chicken gut, proliferating rapidly.
- Eimeria causes avian coccidiosis.
- Eimeria species infecting the gastrointestinal tracts of industrial chickens three species are the primary focus of the invention: E. tenella, E. acervulina, and E. maxima. These are particularly prominent on farms and have different regions of the chicken intestines as invasion target, where they destroy intestinal epithelial cells causing severe damage to the intestinal wall of the chicken.
- An object of the invention is the development of new methods and agents which do not contribute to current antimicrobial resistance.
- the object of the present invention is to provide single-domain antibodies with high binding affinity for the antigen microneme protein 2 (MIC2).
- MIC2 antigens are transmembrane proteins secreted by micronemes to the surface of the anterior region of the parasite in the sporozoite life stage where cell invasion occurs. MIC2 antigens are highly conserved proteins in the Eimeria parasite and are important in multiple interactions, including motility and adherence, between the parasite and the host cell during intestinal epithelial host cell invasion. By blocking the MIC2 antigen, the sporozoite invasion of intestinal epithelial host cells can be inhibited.
- WO2020/234642 discloses polypeptides comprising at least one variable region fragment of a heavy chain antibody (VHH), which specifically binds a disease-causing agent.
- VHH heavy chain antibody
- the object of the present invention is to provide single-domain antibodies with high binding affinity for the antigen microneme protein 2 (MIC2).
- MIC2 antigen microneme protein 2
- the present invention relates to single-domain antibodies having a binding affinity for the antigen microneme protein 2 (MIC2); to polynucleotides encoding the single-domain antibodies; to nucleic acid constructs; to host cells comprising the polynucleotides; to fusion proteins comprising multimer components of the single-domain antibodies; and to the use of the fusion proteins for binding to MIC2 for the prevention of coccidiosis in poultry.
- MIC2 antigen microneme protein 2
- the present invention provides single-domain antibodies (sdAbs) having binding affinity for the antigen microneme protein 2 (MIC2); to fusion proteins comprising two or more sdAbs and to polynucleotides encoding the single-domain antibodies and/or fusion proteins.
- sdAbs single-domain antibodies having binding affinity for the antigen microneme protein 2 (MIC2)
- MIC2 antigen microneme protein 2
- An aspect of the invention is directed to a polypeptide selected from the group consisting of:
- this aspect of the invention is directed to a single-domain antibody having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53.
- the single-domain antibody preferably has binding affinity for the antigen microneme protein 2 (MIC2).
- a second aspect of the invention is directed to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ
- a further aspect of the invention is directed to a single-domain antibody having a binding affinity for the antigen microneme protein 2 (MIC2), such as a binding affinity for one or more of MIC2 from Eimeria tenella (EtMIC2), MIC2 from Eimeria maxima (EmMIC2), and MIC2 from Eimeria acervulina (EaMIC2).
- MIC2 antigen microneme protein 2
- a further aspect of the invention is directed to a single-domain antibody or a fusion protein comprising two or more sdAbs of the invention for preventing, treating or reducing the likelihood of an Eimeria infection in an animal such as poultry, comprising administering to the animal a single-domain antibody or fusion protein of the invention and further comprising a reduced dose or frequency of a coccidostat compared to a conventional dose or frequency.
- a further aspect of the invention is directed to a fusion protein comprising two or more sdAbs of the invention.
- a further aspect of the invention is directed to a composition comprising an sdAb of the invention.
- an aspect of the invention is directed to a composition comprising the fusion protein of the invention.
- the invention further relates to a granule, which comprises: (a) a core comprising the single-domain antibody of the invention or the fusion protein of the invention, and optionally, (b) a coating consisting of one or more layer(s) surrounding the core.
- a related further aspect of the invention is directed to a granule, which comprises: (a) a core, and (b) a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises the singledomain antibody of the invention or the fusion protein of the invention.
- a further aspect is directed to a liquid composition comprising the single-domain antibody of the invention or the fusion protein of the invention, and a stabilizer.
- a key aspect of the invention is directed to use of a fusion protein of the invention or of an sdAb of the invention for the preparation of an animal feed additive or animal feed.
- Related aspects of the invention are directed to a feed additive comprising an sdAb of the invention or the fusion protein of the invention.
- a further related aspect of the invention is directed to a feed comprising a feed additive of the invention.
- a further key aspect of the invention is directed to a method of reducing the likelihood of the spread of Eimeria comprising the use of a granule, composition, feed additive, or feed of the invention.
- a further aspect of the invention is directed to a method of increasing the food conversion ratio (FCR) of an animal or the body weight gain (BWG) of an animal.
- a still further aspect of the invention is directed to a method of treating or preventing coccidiosis in poultry comprising the use of a granule, composition, feed additive, or feed of the invention.
- an aspect of the invention is directed to use of a fusion protein of the invention or an sdAb of the invention for the preparation of a medicament for the prevention or treatment of coccidiosis in poultry, and to a fusion protein of the invention or an sdAb of the invention for use as a medicament, e.g. for the prevention or treatment of coccidiosis in poultry.
- a further aspect of the invention is directed to a method of reducing antimicrobial resistance in animals, such as in poultry farming, said method comprising administering to said animal a feed or feed additive comprising a single-domain antibody or fusion protein of the invention.
- This method of reducing antimicrobial resistance suitably involves blocking binding sites of virulence factors, thereby disabling the virulence factors from binding to host cell receptors, thereby preventing pathogens from establishing an infection in the animals.
- a related aspect is directed to a method of preventing, treating or reducing the likelihood of an Eimeria infection which has a reduced proposentity to induce AMR.
- the invention is furthermore directed to a method or preventing, treating or reducing the likelihood of an Eimeria infection in an animal such as poultry, comprising administering to the animal a single-domain antibody or fusion protein of the invention and further comprising a reduced dose or frequency of a coccidostat compared to a conventional dose or frequency.
- the invention is furthermore directed to a method of reducing parasite invasion or reducing the risk of infections in a pet, such as but not limited to a dog, cat, bird, or rabbit.
- the invention is furthermore directed to a polynucleotide encoding the single-domain antibody of the invention or fusion protein of the invention; to a nucleic acid construct or expression vector comprising the polynucleotide encoding the single-domain antibody of the invention or fusion protein of the invention, operably linked to one or more control sequences that direct the production of the single-domain antibody in an expression host; to a recombinant host cell comprising the nucleic acid construct or expression vector of the invention; and to a method of producing a single-domain antibody or fusion protein, comprising cultivating the recombinant host cell of the invention under conditions conducive for production of the single-domain antibody or fusion protein.
- the Eimeria parasite is the causative agent behind coccidiosis in chickens and is a huge problem in the poultry industry. Low productivity and expenses for treatment and preventive matters are large burdens for farmers. Prevention and treatment with antimicrobial agents have led to increased resistance development against anticoccidial agents in Eimeria species. Fusion proteins comprising single-domain antibodies of the invention bind specifically to virulence factors of pathogenic microorganisms and thereby inactivate their virulence. Fusion proteins comprising single-domain antibodies impose a low selection pressure, do not favour resistance development in the microorganisms, or interact with the beneficial commensals in the gastrointestinal tract as antimicrobial agents do.
- MIC2 is identified for blocking Eimeria adherence and cell invasion.
- Fusion proteins comprising single-domain antibodies of the invention have been constructed to bind specifically to MIC2 and to be mixed with or formulated within animal feed or an animal feed additive. It is alternatively formulated as a coccidiostat product.
- the benefits of the fusion proteins comprising single-domain antibodies of the invention include:
- the fusion proteins comprising single-domain antibodies of the invention are binding proteins suitable as food ingredients and feed additives.
- Figure 2 shows four different binding molecules related to the term ‘antibody’.
- Singledomain antibodies sdAbs
- Fusion proteins are then derived from sdAbs by joining two sdAbs directly or through a linker.
- Figure 3 illustrates the binding affinity of purified sdAbs against target MIC2 antigen (Table 1) evaluated via a standard DELFIA assay.
- TRF means time resolved fluorescence.
- FIG. 5 illustrates the comparison in binding affinity between selected sdAbs and corresponding fusion proteins.
- a standard DELFIA assay was used (Figure 6). Note that the time resolved fluorescence (TRF) signal cannot be compared directly to Figure 3 due to a lower concentration of antibody (50 nM) in this assay.
- TRF time resolved fluorescence
- Figure 6 illustrates a standard DELFIA assay to observe cross-reactivity of fusion proteins (a high concentration (1000 nM))
- Figure 7 illustrates the retained binding affinity of fusion proteins in percentage after treatment with ‘Steam box’, a measurement of thermostability.
- Figure 8 illustrates the binding of fusion proteins to homogenized Eimeria oocysts. Measured with ELISA. Background absorbance for each fusion protein without presence of oocyst was subtracted from each measurement.
- SEQ ID NO:1 is the sequence of a single-domain antibody.
- SEQ ID NO:2 is the sequence of a CDR1 region, found at least in SEQ ID NO:1.
- SEQ ID NO:3 is the sequence of a CDR2 region, found at least in SEQ ID NO:1.
- SEQ ID NO:4 is the sequence of a CDR3 region, found at least in SEQ ID NO:1.
- SEQ ID NO:5 is the sequence of a single-domain antibody.
- SEQ ID NO:6 is the sequence of a CDR1 region, found at least in SEQ ID NO:5.
- SEQ ID NO:7 is the sequence of a CDR2 region, found at least in SEQ ID NO:5.
- SEQ ID NO:8 is the sequence of a CDR3 region, found at least in SEQ ID NO:5.
- SEQ ID NO:9 is the sequence of a single-domain antibody.
- SEQ ID NQ:10 is the sequence of a CDR1 region, found at least in SEQ ID NO:9.
- SEQ ID NO:11 is the sequence of a CDR2 region, found at least in SEQ ID NO:9.
- SEQ ID NO:12 is the sequence of a CDR3 region, found at least in SEQ ID NO:9.
- SEQ ID NO: 13 is the sequence of a single-domain antibody.
- SEQ ID NO:14 is the sequence of a CDR1 region, found at least in SEQ ID NO:13.
- SEQ ID NO:15 is the sequence of a CDR2 region, found at least in SEQ ID NO:13.
- SEQ ID NO: 16 is the sequence of a CDR3 region, found at least in SEQ ID NO: 13.
- SEQ ID NO: 17 is the sequence of a single-domain antibody.
- SEQ ID NO:18 is the sequence of a CDR1 region, found at least in SEQ ID NO:17.
- SEQ ID NO:19 is the sequence of a CDR2 region, found at least in SEQ ID NO:17.
- SEQ ID NQ:20 is the sequence of a CDR3 region, found at least in SEQ ID NO:17.
- SEQ ID NO:21 is the sequence of a single-domain antibody.
- SEQ ID NO:22 is the sequence of a CDR1 region, found at least in SEQ ID NO:21.
- SEQ ID NO:23 is the sequence of a CDR2 region, found at least in SEQ ID NO:21.
- SEQ ID NO:24 is the sequence of a CDR3 region, found at least in SEQ ID NO:21.
- SEQ ID NO:25 is the sequence of a single-domain antibody.
- SEQ ID NO:26 is the sequence of a CDR1 region, found at least in SEQ ID NO:25.
- SEQ ID NO:27 is the sequence of a CDR2 region, found at least in SEQ ID NO:25.
- SEQ ID NO:28 is the sequence of a CDR3 region, found at least in SEQ ID NO:25.
- SEQ ID NO:29 is the sequence of a single-domain antibody.
- SEQ ID NQ:30 is the sequence of a CDR1 region, found at least in SEQ ID NO:29.
- SEQ ID NO:31 is the sequence of a CDR2 region, found at least in SEQ ID NO:29.
- SEQ ID NO:32 is the sequence of a CDR3 region, found at least in SEQ ID NO:29.
- SEQ ID NO:33 is the sequence of a single-domain antibody.
- SEQ ID NO:34 is the sequence of a CDR1 region, found at least in SEQ ID NO:33.
- SEQ ID NO:35 is the sequence of a CDR2 region, found at least in SEQ ID NO:33.
- SEQ ID NO:36 is the sequence of a CDR3 region, found at least in SEQ ID NO:33.
- SEQ ID NO:37 is the sequence of a single-domain antibody.
- SEQ ID NO:38 is the sequence of a CDR1 region, found at least in SEQ ID NO:37.
- SEQ ID NO:39 is the sequence of a CDR2 region, found at least in SEQ ID NO:37.
- SEQ ID NQ:40 is the sequence of a CDR3 region, found at least in SEQ ID NO:37.
- SEQ ID NO:41 is the sequence of a single-domain antibody.
- SEQ ID NO:42 is the sequence of a CDR1 region, found at least in SEQ ID NO:41.
- SEQ ID NO:43 is the sequence of a CDR2 region, found at least in SEQ ID NO:41.
- SEQ ID NO:44 is the sequence of a CDR3 region, found at least in SEQ ID NO:41.
- SEQ ID NO:45 is the sequence of a single-domain antibody.
- SEQ ID NO:46 is the sequence of a CDR1 region, found at least in SEQ ID NO:45.
- SEQ ID NO:47 is the sequence of a CDR2 region, found at least in SEQ ID NO:45.
- SEQ ID NO:48 is the sequence of a CDR3 region, found at least in SEQ ID NO:45.
- SEQ ID NO:49 is the sequence of a single-domain antibody.
- SEQ ID NQ:50 is the sequence of a CDR1 region, found at least in SEQ ID NO:49.
- SEQ ID NO:51 is the sequence of a CDR2 region, found at least in SEQ ID NO:49.
- SEQ ID NO:52 is the sequence of a CDR3 region, found at least in SEQ ID NO:49.
- SEQ ID NO:53 is the sequence of a single-domain antibody.
- SEQ ID NO:54 is the sequence of a CDR1 region, found at least in SEQ ID NO:53.
- SEQ ID NO:55 is the sequence of a CDR2 region, found at least in SEQ ID NO:53.
- SEQ ID NO:56 is the sequence of a CDR3 region, found at least in SEQ ID NO:53.
- SEQ ID NO:57 is the sequence of a CDR3 region of Family 8.
- SEQ ID NO:58 is the sequence of a CDR3 region of Family 8.
- SEQ ID NO:59 is the sequence of a CDR3 region of Family 10.
- SEQ ID NO:60 is the sequence of a CDR3 region of Family 13.
- SEQ ID NO:61 is the sequence of a CDR3 region of Family 13.
- SEQ ID NO:62 is the sequence of a CDR3 region of Family 21.
- SEQ ID NO:63 is the sequence of a CDR3 region of Family 21.
- SEQ ID NO:64 is the sequence of a CDR3 region of Family 21.
- SEQ ID NO:65 is the sequence of a CDR3 region of Family 23.
- SEQ ID NO:66 is the sequence of a CDR3 region of Family 23.
- SEQ ID NO:67 is the sequence of a CDR3 region of Family 23.
- SEQ ID NO:68 is the sequence of a CDR3 region of Family 23.
- SEQ ID NO:69 is the sequence of a CDR3 region of Family 23.
- SEQ ID NQ:70 is the sequence of a CDR3 region of Family 23.
- SEQ ID NO:71 is the sequence of a CDR3 region of Family 23.
- SEQ ID NO:72 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:1 .
- SEQ ID NO:73 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:33.
- SEQ ID NO:74 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:37.
- SEQ ID NO:75 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:45.
- SEQ ID NO:76 is the sequence of a fusion protein compring a homodimer of SEQ ID NO: 13.
- SEQ ID NO:77 is the sequence of a fusion protein compring a homodimer of SEQ ID NO: 17.
- SEQ ID NO:78 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:53.
- SEQ ID NO:79 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:21.
- SEQ ID NQ:80 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:25.
- SEQ ID NO:81 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:9.
- SEQ ID NO:82 is the sequence of a single-domain antibody.
- SEQ ID NO:83 is the sequence of a CDR1 region, found at least in SEQ ID NO:82.
- SEQ ID NO:84 is the sequence of a CDR2 region, found at least in SEQ ID NO:82.
- SEQ ID NO:85 is the sequence of a CDR3 region, found at least in SEQ ID NO:82.
- SEQ ID NO:86 is the sequence of a single-domain antibody.
- SEQ ID NO:87 is the sequence of a CDR1 region, found at least in SEQ ID NO:86.
- SEQ ID NO:88 is the sequence of a CDR2 region, found at least in SEQ ID NO:86.
- SEQ ID NO:89 is the sequence of a CDR3 region, found at least in SEQ ID NO:86.
- SEQ ID NQ:90 is the sequence of a single-domain antibody.
- SEQ ID NO:91 is the sequence of a CDR1 region, found at least in SEQ ID NQ:90.
- SEQ ID NO:92 is the sequence of a CDR2 region, found at least in SEQ ID NQ:90.
- SEQ ID NO:93 is the sequence of a CDR3 region, found at least in SEQ ID NQ:90.
- SEQ ID NO:94 is the sequence of a single-domain antibody.
- SEQ ID NO:95 is the sequence of a CDR1 region, found at least in SEQ ID NO:94.
- SEQ ID NO:96 is the sequence of a CDR2 region, found at least in SEQ ID NO:94.
- SEQ ID NO:97 is the sequence of a CDR3 region, found at least in SEQ ID NO:94.
- SEQ ID NO:98 is the sequence of a single-domain antibody.
- SEQ ID NO:99 is the sequence of a CDR1 region, found at least in SEQ ID NO:98.
- SEQ ID NO: 100 is the sequence of a CDR2 region, found at least in SEQ ID NO:98.
- SEQ ID NQ:101 is the sequence of a CDR3 region, found at least in SEQ ID NO:98.
- SEQ ID NQ:102 is the sequence of a single-domain antibody.
- SEQ ID NQ:103 is the sequence of a CDR1 region, found at least in SEQ ID NQ:102.
- SEQ ID NQ:104 is the sequence of a CDR2 region, found at least in SEQ ID NQ:102.
- SEQ ID NQ:105 is the sequence of a CDR3 region, found at least in SEQ ID NQ:102.
- SEQ ID NQ:106 is the sequence of a single-domain antibody.
- SEQ ID NQ:107 is the sequence of a CDR1 region, found at least in SEQ ID NQ:106.
- SEQ ID NQ:108 is the sequence of a CDR2 region, found at least in SEQ ID NQ:106.
- SEQ ID NQ:109 is the sequence of a CDR3 region, found at least in SEQ ID NQ:106.
- SEQ ID NQ:110 is the sequence of a single-domain antibody.
- SEQ ID NO:111 is the sequence of a CDR1 region, found at least in SEQ ID NQ:110.
- SEQ ID NO:112 is the sequence of a CDR2 region, found at least in SEQ ID NQ:110.
- SEQ ID NO:113 is the sequence of a CDR3 region, found at least in SEQ ID NQ:110.
- SEQ ID NO:114 is the sequence of a single-domain antibody.
- SEQ ID NO:115 is the sequence of a CDR1 region, found at least in SEQ ID NO:114.
- SEQ ID NO:116 is the sequence of a CDR2 region, found at least in SEQ ID NO:114.
- SEQ ID NO:117 is the sequence of a CDR3 region, found at least in SEQ ID NO:114.
- SEQ ID NO:118 is the sequence of a single-domain antibody.
- SEQ ID NO:119 is the sequence of a CDR1 region, found at least in SEQ ID NO:118.
- SEQ ID NQ:120 is the sequence of a CDR2 region, found at least in SEQ ID NO:118.
- SEQ ID NO:121 is the sequence of a CDR3 region, found at least in SEQ ID NO:118.
- SEQ ID NO:122 is the sequence of a single-domain antibody.
- SEQ ID NO:123 is the sequence of a CDR1 region, found at least in SEQ ID NO:122.
- SEQ ID NO:124 is the sequence of a CDR2 region, found at least in SEQ ID NO:122.
- SEQ ID NO:125 is the sequence of a CDR3 region, found at least in SEQ ID NO:122.
- SEQ ID NO: 126 is the sequence of a single-domain antibody.
- SEQ ID NO:127 is the sequence of a CDR1 region, found at least in SEQ ID NO:126.
- SEQ ID NO:128 is the sequence of a CDR2 region, found at least in SEQ ID NO:126.
- SEQ ID NO:129 is the sequence of a CDR3 region, found at least in SEQ ID NO:126.
- SEQ ID NQ:130 is the sequence of a single-domain antibody.
- SEQ ID NO:131 is the sequence of a CDR1 region, found at least in SEQ ID NQ:130.
- SEQ ID NO:132 is the sequence of a CDR2 region, found at least in SEQ ID NQ:130.
- SEQ ID NO:133 is the sequence of a CDR3 region, found at least in SEQ ID NQ:130.
- SEQ ID NO: 134 is the sequence of a single-domain antibody.
- SEQ ID NO:135 is the sequence of a CDR1 region, found at least in SEQ ID NO:134.
- SEQ ID NO:136 is the sequence of a CDR2 region, found at least in SEQ ID NO:134.
- SEQ ID NO:137 is the sequence of a CDR3 region, found at least in SEQ ID NO:134.
- SEQ ID NO: 138 is the sequence of a single-domain antibody.
- SEQ ID NO:139 is the sequence of a CDR1 region, found at least in SEQ ID NO:138.
- SEQ ID NQ:140 is the sequence of a CDR2 region, found at least in SEQ ID NO:138.
- SEQ ID NO:141 is the sequence of a CDR3 region, found at least in SEQ ID NO:138.
- SEQ ID NO:142 is the sequence of a single-domain antibody.
- SEQ ID NO:143 is the sequence of a CDR1 region, found at least in SEQ ID NO:142.
- SEQ ID NO:144 is the sequence of a CDR2 region, found at least in SEQ ID NO:142.
- SEQ ID NO:145 is the sequence of a CDR3 region, found at least in SEQ ID NO:142.
- SEQ ID NO:146 is the sequence of a single-domain antibody.
- SEQ ID NO:147 is the sequence of a CDR1 region, found at least in SEQ ID NO:146.
- SEQ ID NO:148 is the sequence of a CDR2 region, found at least in SEQ ID NO:146.
- SEQ ID NO:149 is the sequence of a CDR3 region, found at least in SEQ ID NO:146.
- SEQ ID NO: 150 is the sequence of a single-domain antibody.
- SEQ ID NO:151 is the sequence of a CDR1 region, found at least in SEQ ID NQ:150.
- SEQ ID NO:152 is the sequence of a CDR2 region, found at least in SEQ ID NQ:150.
- SEQ ID NO:153 is the sequence of a CDR3 region, found at least in SEQ ID NQ:150.
- SEQ ID NO: 154 is the sequence of a single-domain antibody.
- SEQ ID NO:155 is the sequence of a CDR1 region, found at least in SEQ ID NO:154.
- SEQ ID NO:156 is the sequence of a CDR2 region, found at least in SEQ ID NO:154.
- SEQ ID NO:157 is the sequence of a CDR3 region, found at least in SEQ ID NO:154.
- SEQ ID NO: 158 is the sequence of a single-domain antibody.
- SEQ ID NO:159 is the sequence of a CDR1 region, found at least in SEQ ID NO:158.
- SEQ ID NQ:160 is the sequence of a CDR2 region, found at least in SEQ ID NO:158.
- SEQ ID NO:161 is the sequence of a CDR3 region, found at least in SEQ ID NO:158.
- SEQ ID NO:162 is the sequence of a single-domain antibody.
- SEQ ID NO:163 is the sequence of a CDR1 region, found at least in SEQ ID NO:162.
- SEQ ID NO:164 is the sequence of a CDR2 region, found at least in SEQ ID NO:162.
- SEQ ID NO:165 is the sequence of a CDR3 region, found at least in SEQ ID NO:162.
- SEQ ID NO:166 is the sequence of a single-domain antibody.
- SEQ ID NO:167 is the sequence of a CDR1 region, found at least in SEQ ID NO:166.
- SEQ ID NO:168 is the sequence of a CDR2 region, found at least in SEQ ID NO:166.
- SEQ ID NO:169 is the sequence of a CDR3 region, found at least in SEQ ID NO:166.
- SEQ ID NO: 170 is the sequence of a single-domain antibody.
- SEQ ID NO:171 is the sequence of a CDR1 region, found at least in SEQ ID NQ:170.
- SEQ ID NO:172 is the sequence of a CDR2 region, found at least in SEQ ID NQ:170.
- SEQ ID NO:173 is the sequence of a CDR3 region, found at least in SEQ ID NQ:170.
- SEQ ID NO: 174 is the sequence of a single-domain antibody.
- SEQ ID NO:175 is the sequence of a CDR1 region, found at least in SEQ ID NO:174.
- SEQ ID NO:176 is the sequence of a CDR2 region, found at least in SEQ ID NO:174.
- SEQ ID NO:177 is the sequence of a CDR3 region, found at least in SEQ ID NO:174.
- SEQ ID NO:178 is the sequence of a single-domain antibody.
- SEQ ID NO:179 is the sequence of a CDR1 region, found at least in SEQ ID NO:178.
- SEQ ID NQ:180 is the sequence of a CDR2 region, found at least in SEQ ID NO:178.
- SEQ ID NO:181 is the sequence of a CDR3 region, found at least in SEQ ID NO:178.
- SEQ ID NO:182 is the sequence of a single-domain antibody.
- SEQ ID NO:183 is the sequence of a CDR1 region, found at least in SEQ ID NO:182.
- SEQ ID NO:184 is the sequence of a CDR2 region, found at least in SEQ ID NO:182.
- SEQ ID NO:185 is the sequence of a CDR3 region, found at least in SEQ ID NO:182.
- SEQ ID NO:186 is the sequence of a single-domain antibody.
- SEQ ID NO:187 is the sequence of a CDR1 region, found at least in SEQ ID NO:186.
- SEQ ID NO:188 is the sequence of a CDR2 region, found at least in SEQ ID NO:186.
- SEQ ID NO:189 is the sequence of a CDR3 region, found at least in SEQ ID NO:186.
- SEQ ID NQ:190 is the sequence of a single-domain antibody.
- SEQ ID NO:191 is the sequence of a CDR1 region, found at least in SEQ ID NQ:190.
- SEQ ID NO:192 is the sequence of a CDR2 region, found at least in SEQ ID NQ:190.
- SEQ ID NO:193 is the sequence of a CDR3 region, found at least in SEQ ID NQ:190.
- SEQ ID NO:194 is the sequence of a single-domain antibody.
- SEQ ID NO:195 is the sequence of a CDR1 region, found at least in SEQ ID NO:194.
- SEQ ID NO:196 is the sequence of a CDR2 region, found at least in SEQ ID NO:194.
- SEQ ID NO:197 is the sequence of a CDR3 region, found at least in SEQ ID NO:194.
- SEQ ID NO:198 is the sequence of a single-domain antibody.
- SEQ ID NO:199 is the sequence of a CDR1 region, found at least in SEQ ID NO:198.
- SEQ ID NQ:200 is the sequence of a CDR2 region, found at least in SEQ ID NO:198.
- SEQ ID NQ:201 is the sequence of a CDR3 region, found at least in SEQ ID NO:198.
- SEQ ID NQ:202 is the sequence of a single-domain antibody.
- SEQ ID NQ:203 is the sequence of a CDR1 region, found at least in SEQ ID NQ:202.
- SEQ ID NQ:204 is the sequence of a CDR2 region, found at least in SEQ ID NQ:202.
- SEQ ID NQ:205 is the sequence of a CDR3 region, found at least in SEQ ID NQ:202.
- SEQ ID NQ:206 is the sequence of a single-domain antibody.
- SEQ ID NQ:207 is the sequence of a CDR1 region, found at least in SEQ ID NQ:206.
- SEQ ID NQ:208 is the sequence of a CDR2 region, found at least in SEQ ID NQ:206.
- SEQ ID NQ:209 is the sequence of a CDR3 region, found at least in SEQ ID NQ:206.
- SEQ ID NQ:210 is the sequence of a single-domain antibody.
- SEQ ID NO:211 is the sequence of a CDR1 region, found at least in SEQ ID NQ:210.
- SEQ ID NO:212 is the sequence of a CDR2 region, found at least in SEQ ID NO:210.
- SEQ ID NO:213 is the sequence of a CDR3 region, found at least in SEQ ID NO:210.
- SEQ ID NO:214 is the sequence of a single-domain antibody.
- SEQ ID NO:215 is the sequence of a CDR1 region, found at least in SEQ ID NO:214.
- SEQ ID NO:216 is the sequence of a CDR2 region, found at least in SEQ ID NO:214.
- SEQ ID NO:217 is the sequence of a CDR3 region, found at least in SEQ ID NO:214.
- SEQ ID NO:218 is the sequence of a single-domain antibody.
- SEQ ID NO:219 is the sequence of a CDR1 region, found at least in SEQ ID NO:218.
- SEQ ID NQ:220 is the sequence of a CDR2 region, found at least in SEQ ID NO:218.
- SEQ ID NO:221 is the sequence of a CDR3 region, found at least in SEQ ID NO:218.
- SEQ ID NO:222 is the sequence of a single-domain antibody.
- SEQ ID NO:223 is the sequence of a CDR1 region, found at least in SEQ ID NO:222.
- SEQ ID NO:224 is the sequence of a CDR2 region, found at least in SEQ ID NO:222.
- SEQ ID NO:225 is the sequence of a CDR3 region, found at least in SEQ ID NO:222.
- SEQ ID NO:226 is the sequence of a single-domain antibody.
- SEQ ID NO:227 is the sequence of a CDR1 region, found at least in SEQ ID NO:226.
- SEQ ID NO:228 is the sequence of a CDR2 region, found at least in SEQ ID NO:226.
- SEQ ID NO:229 is the sequence of a CDR3 region, found at least in SEQ ID NO:226.
- SEQ ID NQ:230 is the sequence of a single-domain antibody.
- SEQ ID NO:231 is the sequence of a CDR1 region, found at least in SEQ ID NQ:230.
- SEQ ID NO:232 is the sequence of a CDR2 region, found at least in SEQ ID NQ:230.
- SEQ ID NO:233 is the sequence of a CDR3 region, found at least in SEQ ID NQ:230.
- SEQ ID NO:234 is the sequence of a single-domain antibody.
- SEQ ID NO:235 is the sequence of a CDR1 region, found at least in SEQ ID NO:234.
- SEQ ID NO:236 is the sequence of a CDR2 region, found at least in SEQ ID NO:234.
- SEQ ID NO:237 is the sequence of a CDR3 region, found at least in SEQ ID NO:234.
- SEQ ID NO:238 is the sequence of a single-domain antibody.
- SEQ ID NO:239 is the sequence of a CDR1 region, found at least in SEQ ID NO:238.
- SEQ ID NQ:240 is the sequence of a CDR2 region, found at least in SEQ ID NO:238.
- SEQ ID NO:241 is the sequence of a CDR3 region, found at least in SEQ ID NO:238.
- SEQ ID NO:242 is the sequence of a single-domain antibody.
- SEQ ID NO:243 is the sequence of a CDR1 region, found at least in SEQ ID NO:242.
- SEQ ID NO:244 is the sequence of a CDR2 region, found at least in SEQ ID NO:242.
- SEQ ID NO:245 is the sequence of a CDR3 region, found at least in SEQ ID NO:242. Definitions
- Antibody commonly refers to large human antibodies. According to the invention, the smaller single-domain antibodies (sdAbs) have beneficial biochemical and cost of manufacturing properties. Single-domain antibodies, also known as ‘nanobodies’ or ‘VHHs’, are small binding molecules derived from camelid antibodies.
- Animal refers to all animals except humans. Examples of animals are non-ruminants, and ruminants. Ruminant animals include, for example, animals such as sheep, goats, cattle, e.g. beef cattle, cows, and young calves, deer, yank, camel, llama and kangaroo. Non-ruminant animals include mono-gastric animals, e.g.
- pigs or swine including, but not limited to, piglets, growing pigs, and sows
- poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); cats; dogs; horses (including but not limited to hotbloods, coldbloods and warm bloods), young calves; fish (including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sau
- Animal feed refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal.
- Animal feed for a monogastric animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbial, amino acids, and/or other feed ingredients (such as in a premix) whereas animal feed for ruminants generally comprises forage (including roughage and silage) and may further comprise concentrates as well as vitamins, minerals, enzymes direct fed microbial, amino acid, and/or other feed ingredients (such as in a premix).
- Concentrates means feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from e.g. corn, oats, rye, barley, wheat), oilseed press cake (e.g. from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).
- high protein and energy concentrations such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from e.g. corn, oats, rye, barley, wheat), oilseed press cake (e.g. from cottonseed, safflower, sunflower, soybean (such as soybean meal
- cDNA means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA.
- the initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
- CDR region means complementarity-determining region (in sdAbs there are three: CDR1 , CDR2 and CDR3).
- the CDR regions are part of the variable domain of an antibody, including the sdAbs of the present invention, and determine the antigen-binding specificity of the antibody.
- the CDR3 is the most variable of the CDRs and is a key determinant of antibody binding specificity.
- Coding sequence means a polynucleotide, which directly specifies the amino acid sequence of a single-domain antibody.
- the boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA.
- the coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
- control sequences means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native (/.e., from the same gene) or heterologous (/.e., from a different gene) to the polynucleotide encoding the single-domain antibody, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a single-domain antibody.
- Expression means any step involved in the production of a singledomain antibody including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
- Expression vector refers to a linear or circular DNA construct comprising a DNA sequence encoding a single-domain antibody, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host.
- control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.
- Forage refers to sdAbs as classified by their CDR3 homologies.
- Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof.
- forage plants are Alfalfa (lucerne), birdsfoot trefoil, brassica (e.g. kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g. alsike clover, red clover, subterranean clover, white clover), grass (e.g.
- Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains), residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.
- Fusion peptide is a singledomain antibody in which a first single-domain antibody is fused at the N-terminus and/or the C-terminus with a second single-domain antibody of the present invention.
- a fusion single-domain antibody may be produced by fusing a polynucleotide encoding a singledomain antibody of the present invention to a polynucleotide encoding another polypeptide, or by fusing two or more polynucleotides of the present invention together. Otherwise stated, the process involves extending one sdAbs coding sequences with the coding sequences of one or more polypeptides.
- Techniques for producing fusion single-domain antibodies include ligating the coding sequences encoding the single-domain antibodies so that they are in frame and that expression of the fusion single-domain antibody is under control of the same promoter(s) and terminator.
- Fusion single-domain antibodies may also be constructed using intein technology in which fusion single-domain antibodies are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).
- a fusion single-domain antibody can further comprise a cleavage site between the two single-domain antibodies. Upon secretion of the fusion protein, the site is cleaved releasing the two single-domain antibodies. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J.
- homodimer means a homo divalent protein construct and refers to a protein comprising a first peptide having a first binding specificity and a second peptide having a second binding specificity connected by a linker, wherein the peptides are the same or substantially the same such as having at least 70% sequence identity.
- Heterodimer means a hetero divalent protein construct and refers to a protein comprising a first peptide having a first binding specificity and a second peptide having a second binding specificity connected by a linker, wherein the peptides are different or substantially different such as having less than 70% sequence identity.
- Heterologous means, with respect to a host cell, that a singledomain antibody or nucleic acid does not naturally occur in the host cell.
- heterologous means, with respect to a single-domain antibody or nucleic acid, that a control sequence, e.g., promoter, of a single-domain antibody or nucleic acid is not naturally associated with the singledomain antibody or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature single-domain antibody.
- Host Strain or Host Cell is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a single-domain antibody of the present invention has been introduced.
- Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the single-domain antibody of interest and/or fermenting saccharides.
- the term "host cell” includes protoplasts created from cells.
- Improving means that one or more parameters influenced by an infection, such as Eimeria and Clostridium perfringens infections, are changing in a desireable direction. For example, in the broiler chickens with Eimeria and Clostridium perfringens infections, the body weight gain is increased, the body lesion is reduced and/or the expression of the antiinflammatory cytokines such as IL10 and IL8 is increased, etc.
- Isolated means a single-domain antibody, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc.
- An isolated singledomain antibody, nucleic acid, cell, or other material is thus in a form that does not occur in nature.
- An isolated single-domain antibody includes, but is not limited to, a culture broth containing the secreted single-domain antibody expressed in a host cell.
- Mature single-domain antibody means a single-domain antibody in its mature form following N-terminal and/or C-terminal processing (e.g., removal of signal peptide).
- Mature single-domain antibody coding sequence means a polynucleotide that encodes a mature single-domain antibody having MIC2 binding affinity.
- MIC2 means the antigen microneme protein 2 from Eimeria, more precisely the Microneme protein 2 of the Eimeria spp. genus or equivalent homologs from other apicomplexan parasites, such as the MIC2-associated protein (M2AP) of the Toxoplasma spp. genus.
- Figure 1 shows the alignment of MIC2 amino acid sequences.
- Native means a nucleic acid or single-domain antibody naturally occurring in a host cell.
- Nucleic acid encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a single-domain antibody. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerated, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.
- nucleic acid construct means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.
- operably linked means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner.
- a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.
- preventing means the prevention of the onset, recurrence, or spread, in whole or in part, of an infection such as Eimeria and Clostridium perfringens infections, or a symptom thereof.
- purified means a nucleic acid, single-domain antibody, or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified single-domain antibody or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation).
- a purified nucleic acid or single-domain antibody is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis).
- a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique.
- the term "enriched" refers to a compound, single-domain antibody, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.
- the term “purified” as used herein refers to the single-domain antibody or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects the term “purified” refers to the single-domain antibody being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the single-domain antibody is separated from some of the soluble components of the organism and culture medium from which it is recovered. The single-domain antibody may be purified (/.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.
- the single-domain antibody may be purified such that only minor amounts of other proteins, in particular other single-domain antibodies, are present.
- the term "purified” as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the single-domain antibody.
- the singledomain antibody may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced single-domain antibody.
- the single-domain antibody is at least 40% pure by weight of the total singledomain antibody material present in the preparation.
- the single-domain antibody is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total single-domain antibody material present in the preparation.
- substantially pure single-domain antibody may denote a single-domain antibody preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1 %, and even most preferably at most 0.5% by weight of other single-domain antibody material with which the single-domain antibody is natively or recombinantly associated.
- the substantially pure single-domain antibody is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total singledomain antibody material present in the preparation.
- the single-domain antibody of the present invention is preferably in a substantially pure form ⁇ i.e., the preparation is essentially free of other single-domain antibody material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the single-domain antibody by well-known recombinant methods or by classical purification methods.
- Recombinant is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature.
- the term recombinant refers to a cell, nucleic acid, single-domain antibody or vector that has been modified from its native state.
- recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature.
- the term “recombinant” is synonymous with “genetically modified” and “transgenic”.
- Recover means the removal of a single-domain antibody from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the single-domain antibody from the whole fermentation broth, or from the cell-free fermentation broth, by single-domain antibody crystal harvest, by filtration, e.g., depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheed or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hyrdo cyclones or similar), or by precipitating the single-domain antibody and using relevant solid-liquid separation methods to
- Roughage means dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste).
- Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.
- the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later.
- the parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
- the Needle program In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line.
- the output of Needle labeled “longest identity” is calculated as follows:
- the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later.
- the parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix.
- the nobrief option must be specified in the command line.
- the output of Needle labeled “longest identity” is calculated as follows:
- Signal Peptide A "signal peptide” is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell.
- the mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.
- Treating means alleviation, in whole or in part, of an infection, such as Eimeria and Clostridium perfringens infections, or a symptom thereof, or slowing, or halting of further progression or worsening of an infection.
- variant means a single-domain antibody having MIC2 binding activity comprising a man-made mutation, i.e., a substitution, insertion (including extension), and/or deletion (e.g., truncation), at one or more positions.
- a substitution means replacement of the amino acid occupying a position with a different amino acid;
- a deletion means removal of the amino acid occupying a position; and
- an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.
- Antibody engineering is a powerful technique to improve the effector function, stability, or utilize the binding abilities of antibodies in different contexts.
- camelids camels, llamas, alpacas etc.
- the camelid antibodies consist of two identical polypeptide chains each containing two constant domains (CH2 and CH3) and a single variable domain ( HH).
- the molecule is 2/3 the size of a human monoclonal antibody (mAb).
- the single-domain antibody is the recombinant variable domain ( HH) of camelid heavy-chain antibodies (hcAb), and, said more generally, a small stable binding protein.
- sdAbs have a unique structure and several advantageous functions compared to many other binding proteins and antibody formats, including high stability, high specificity, and affinity, good solubility, and low immunogenicity. Additionally, the relatively small size of 12-17 kD allows sdAbs to act in narrow or hidden regions where a human mAb would not fit. sdAbs have high stability and are very robust compared to many other proteins. They can resist chemical and thermal denaturation. sdAbs have the capacity of reversible refolding, which make them able to return to physiological conditions. They can be stored for months at 4 °C and even longer at -20°C and still maintain the antigen-binding capacity.
- sdAbs have good solubility since they do not have a hydrophobic interaction, as human mAbs have, instead they have a hydrophilic side.
- the low production costs and beneficial biochemical properties of sdAbs make them ideal candidates as sustainable functional food ingredients and feed additives.
- the fusion protein products are constructed of two sdAbs, such as two identical sdAbs, linked together by a protein linker, increasing their functional binding affinity due to increased avidity.
- the fusion proteins of the invention can be ingested orally as a feed additive because of the high stability of the product, combined with being active and binding extracellularly to virulence factors throughout the gastrointestinal tract. Ingestion of the fusion proteins of the invention reduces the risk of gastrointestinal infections and supports a healthy gut microbiome.
- the invention is directed to single-domain antibodies having a binding affinity for the antigen microneme protein 2 (MIC2A).
- the invention is directed to a single-domain antibody comprising a CDR3 region having at least 75% seguence identity to a seguence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28,
- SEQ ID NO:36 SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:58,
- the single-domain antibody comprises a CDR3 region having at least 80% seguence identity, such as at least 85% seguence identity, or at least 90% seguence identity, such as at least 95% seguence identity, such as 100% seguence identity, to a seguence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44,
- SEQ ID NO:62 SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,
- the CDR3 region has at least 75% seguence identity, such as at least 80% seguence identity, such as at least 85% seguence identity, or at least 90% seguence identity, such as at least 95% seguence identity, such as 100% seguence identity, to a seguence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, and SEQ ID NO:56.
- the CDR3 region has at least 75% seguence identity, such as at least 80% seguence identity, such as at least 85% seguence identity, or at least 90% seguence identity, such as at least 95% seguence identity, such as 100% seguence identity, to a seguence selected from the group consisting of SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:48 and SEQ ID NO:56.
- the single-domain antibody comprises a CDR3 region having a sequence selected from the group consisting ofSEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44,
- SEQ ID NO:62 SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,
- SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70 and SEQ ID NO:71 such as a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, and SEQ ID NO:56, typically a sequence selected from the group consisting of SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:48 and SEQ ID NO:56.
- the inventors have identified a number of unique sdAbs, which can be categorized according to their CDR3 regions.
- the sdAb comprising SEQ ID NO: 16 is categorized in Family 8 where there is high homology within the CDR3 region of the sdAb. These include SEQ ID NO: 57 and SEQ ID NO: 58.
- the sdAb comprises a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 57 and SEQ ID NO: 58.
- the CDR3 region has as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 57 and SEQ ID NO: 58.
- the sdAb comprises a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO: 59.
- the CDR3 region has as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO: 59.
- a further family of interest are sdAbs of Family 13, which comprises SEQ ID NO:28, SEQ ID NQ:60 and SEQ ID NO: 61.
- the sdAb comprises a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NQ:60 and SEQ ID NO: 61 .
- the CDR3 region has as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NQ:60 and SEQ ID NO: 61.
- a further family of interest are sdAbs of Family 21 , which comprises SEQ ID NQ:20, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO: 64.
- the sdAb comprises a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO: 64.
- the CDR3 region has as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO: 64.
- a further family of interest are sdAbs of Family 23, which comprises SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70 and SEQ ID NO:71 .
- the sdAb comprises a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71 .
- the CDR3 region has as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71.
- the sdAb comprises a CDR3 region from one of the identified preferred families, namely from any one of Family 8, 10, 13, 21 and 23, such as having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:24, SEQ ID NO: 59, SEQ ID NO:28, SEQ ID NQ:60, SEQ ID NO: 61 , SEQ ID NQ:20, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71.
- the CDR3 region has as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 57, SEQ ID NO: 58 SEQ ID NO:24, SEQ ID NO: 59, SEQ ID NO:28, SEQ ID NQ:60, SEQ ID NO: 61 , SEQ ID NQ:20, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71.
- the single-domain antibody defined by its CDR3 region can furthermore be defined as comprising a CDR1 region.
- the single-domain antibody of the invention may comprise a CDR3 as defined herein and further comprise a CDR1 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NQ:30, SEQ ID NO:42, SEQ ID NQ:50, SEQ ID NO:54, SEQ ID NQ:107, SEQ ID NO:111 , SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:147, SEQ ID NO:159, SEQ ID NO:163, SEQ ID NO:183, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:199, SEQ ID NO:211 , SEQ ID NO:215 and SEQ ID NO:239.
- the single-domain antibody defined by its CDR3 region can furthermore be defined as comprising a CDR2 region.
- the single-domain antibody of the invention may comprise a CDR3 as defined herein and further comprise a CDR2 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:39, SEQ ID NO:51 , SEQ ID NO:136, SEQ ID NO:164, SEQ ID NO:172, SEQ ID NQ:180, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:192, SEQ ID NO:212 and SEQ ID NO:236.
- the single-domain antibody defined by its CDR3 region can furthermore be defined as further comprising a CDR1 region and a CDR2 region.
- the single-domain antibody of the invention may comprise a CDR3 as defined herein and further comprise a CDR1 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NQ:30, SEQ ID NO:42, SEQ ID NQ:50, SEQ ID NO:54, SEQ ID NQ:107, SEQ ID NO:111 , SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:147, SEQ ID NO:159, SEQ ID NO:163, SEQ ID NO:183, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:199, SEQ ID NO:211 , SEQ ID NO:215 and SEQ ID NO:2
- the single-domain antibody is selected from the group comprising a. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:2, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 3, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 4; b. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:6, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 7, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 8; c.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 10, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 11 , and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 12; d. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 14, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 15, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:16; e.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 18, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 19, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 20; f. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:22, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 23, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 24 g.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:26, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 27, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 28; h. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NQ:30, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 31 , and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 32; i.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:34, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 35, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 36; j. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:38, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 39, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 40; k.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:42, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 43, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 44; l. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:46, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 47, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 48; m.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NQ:50, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 51 , and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 52; n. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:54, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 55, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 56; o.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:83, a CDR2 region having at least 75% sequence identity to SEQ ID NO:84, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:85;
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:87, a CDR2 region having at least 75% sequence identity to SEQ ID NO:88, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:89;
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:91 , a CDR2 region having at least 75% sequence identity to SEQ ID NO:92, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:93; r. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:95, a CDR2 region having at least 75% sequence identity to SEQ ID NO:96, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:97; s.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:99, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:100, and a CDR3 region having at least 75% sequence identity to SEQ ID NQ:101 ;
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 103, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 104, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 105; u.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 107, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 108, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 109;
- v. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 111 , a CDR2 region having at least 75% sequence identity to SEQ ID NO: 112, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 113; w.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 115, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 116, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 117; x. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 119, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 120, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 121 ; y.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 123, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 124, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 125; z. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 127, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 128, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 129; aa.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:131 , a CDR2 region having at least 75% sequence identity to SEQ ID NO: 132, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 133; bb. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 135, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 136, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 137; cc.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 139, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:140, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:141 ; dd. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 143, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 144, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 145; ee.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 147, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 148, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 149; ff. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:151 , a CDR2 region having at least 75% sequence identity to SEQ ID NO: 152, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 153; gg.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 155, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 156, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 157; hh. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 159, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:160, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:161 ; ii.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 163, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 164, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 165; jj. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 167, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 168, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 169; kk.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 171 , a CDR2 region having at least 75% sequence identity to SEQ ID NO: 172, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 173;
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 175, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 176, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 177;
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 179, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 180, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:181 ; nn.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 183, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 184, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 185; oo. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 187, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 188, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 189; pp.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:191 , a CDR2 region having at least 75% sequence identity to SEQ ID NO: 192, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 193; qq. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 195, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 196, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 197; rr.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 199, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:200, and a CDR3 region having at least 75% sequence identity to SEQ ID NQ:201 ; ss. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NQ:203, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:204, and a CDR3 region having at least 75% sequence identity to SEQ ID NQ:205; tt.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NQ:207, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:208, and a CDR3 region having at least 75% sequence identity to SEQ ID NQ:209; uu. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:211 , a CDR2 region having at least 75% sequence identity to SEQ ID NO:212, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:213; vv.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:215, a CDR2 region having at least 75% sequence identity to SEQ ID NO:216, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:217; ww. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:219, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:220, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:221 ; xx.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:223, a CDR2 region having at least 75% sequence identity to SEQ ID NO:224, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:225; yy. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:227, a CDR2 region having at least 75% sequence identity to SEQ ID NO:228, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:229; zz.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:231 , a CDR2 region having at least 75% sequence identity to SEQ ID NO:232, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:233; aaa. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:235, a CDR2 region having at least 75% sequence identity to SEQ ID NO:236, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:237; bbb.
- a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:239, a CDR2 region having at least 75% sequence identity to SEQ ID NO:240, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:241 ; and ccc. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:243, a CDR2 region having at least 75% sequence identity to SEQ ID NO:244, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:245.
- a single-domain antibody has at least 80% sequence identity, such as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96, 97, 98, or 99% sequence identity, to a sequence of the invention.
- the invention is directed to a single-domain antibody having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO
- a single-domain antibody has at least 80% sequence identity, such as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96, 97, 98, or 99% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122
- the single-domain antibody comprises, consists essentially of, or consists of a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, preferably SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:
- a further aspect of the invention is directed to a single-domain antibody comprising a CDR3 region having a binding affinity for the antigen microneme protein 2 (MIC2), such as a binding affinity for one or more of MIC2 from Eimeria tenella (EtMIC2), MIC2 from Eimeria maxima (EmMIC2), and MIC2 from Eimeria acervulina (EaMIC2).
- the single-domain antibody may comprise as CDR3 is as defined by the invention.
- an object of the invention is an sdAb which is cross-reactive, binding more than one MIC2 variant.
- a further object of the invention is an sdAb that only have specificity against single MIC2 variants.
- An immunization strategy where all three variants were used for co-immunization increased the likelihood of cross-reactive sdAbs.
- the single-domain antibody has a Kd for EaMIC2 of 10 nM or less.
- the single-domain antibody has a Kd for EaMIC2 of 10 nM or less and has a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO: 59 of Family 10 and SEQ ID NO:28, SEQ ID NQ:60 and SEQ ID NO: 61 of Family 13.
- the single-domain has a Kd for EaMIC2 of 8 nM or less, such as 7, 6, 5, 4 3, 2, or 1 nM or less and the CDR3 region has at least 85% sequence identity or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO: 59 of Family 10 and SEQ ID NO:28, SEQ ID NO:60 and SEQ ID NO: 61 of Family 13
- the single-domain antibody has a Kd for EmMIC2 of 10 nM or less.
- the single-domain antibody has a Kd for for EmMIC2 of 10 nM or less and has at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 57 and SEQ ID NO: 58 of Family 8 and SEQ ID NQ:20, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO: 64 of Family 21.
- the singledomain has a Kd for EmMIC2 of 8 nM or less, such as 7, 6, 5, 4 3, 2, or 1 nM or less and the CDR3 region has at least 85% sequence identity or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 57 and SEQ ID NO: 58 of Family 8 and SEQ ID NQ:20, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO: 64 of Family 21 .
- the single-domain antibody has cross-reactivity by having a binding affinity to at least two of EaMIC2, EmMIC2, and EtMIC2 wherein it has a binding affinity with a Kd of 10 nM or less, at least one of EaMIC2, EmMIC2 and EtMIC2.
- the single-domain antibody has a cross-reactivity by binding to at least two of EaMIC2, EmMIC2, and EtMIC2 with a Kd of 10 nM or less, preferably having a cross-reactivity by binding to all three of EaMIC2, EmMIC2, and EtMIC2 with a Kd of 10 nM or less and wherein the CDR3 region has at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71 of Family 23.
- the single-domain antibody has a cross-reactivity by binding to at least two of EaMIC2, EmMIC2, and EtMIC2 with a Kd of 8 nM or less, such as 7, 6, 5, 4 3, 2, or 1 nM or less and the CDR3 region has at least 85% sequence identity or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity to a sequence selected from the group consisting of of SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71 of Family 23.
- the single-domain antibody has cross-reactivity in having a binding affinity to at least two of EaMIC2, EmMIC2, and EtMIC2 with a Kd of 10 nM or less to at least one of EaMIC2, EmMIC2, and EtMIC2 and further comprises a CDR1 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NQ:30, SEQ ID NO:42, SEQ ID NQ:50, SEQ ID NO:54, SEQ ID NQ:107, SEQ ID NO:111 , SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:147, SEQ ID NO:159, SEQ ID NO:163, SEQ ID NO:183, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:199, SEQ ID NO:211 , SEQ ID NO:
- the single-domain antibody has cross-reactivity by having a binding affinity to at least two of EaMIC2, EmMIC2, and EtMIC2 and furthermore having a Kd of 10 nM or less to at least one of EaMIC2, EmMIC2, and EtMIC2 and may further comprise a CDR2 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:39, SEQ ID NO:51 , SEQ ID NO:136, SEQ ID NO:164, SEQ ID NO:172, SEQ ID NQ:180, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:192, SEQ ID NO:212 and SEQ ID NO:236.
- the single-domain antibody has cross-reactivity by having a binding affinity to at least two of EaMIC2, EmMIC2, and EtMIC2 and having a Kd of 10 nM or less to at least one of EaMIC2, EmMIC2, and EtMIC2 and may further have at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, preferably SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49
- sdAbs targeting EtMIC2 were highly similar in sequence. Particularly interesting is the highly similar CDR3 region. Previously, it was noted that these sdAbs also displayed a similar TRF (time resolved fluorescence) signal in a DELFIA assay, which underlines the similarity. This means that sdAbs with high affinity to EtMIC2 in our hands are highly conserved and therefore subject to optimization. Based on thorough review of affinity and sequence variation, sdAbs were selected for homodimer construction (Table 1). Emphasis was put on proven high affinity, both during ranking and for purified protein, as well as sequence diversity.
- Single-domain antibodies of the invention can be obtained in a method comprising the use of camels or alpacas.
- alpacas were immunized with MIC2 antigens.
- the animals were injected with antigen a total of 5 times over a 35-day period. From each animal, blood was then collected.
- RNA was prepared from the blood, pooled, and used for cDNA synthesis.
- sdAb genes could be PCR amplified before being cloned into a phagemid vector. In this way, core libraries containing approximately 1 .8x10 9 genes of the correct size were generated. These libraries were panned, using phage display, over solid-phase coated MIC2 antigen for a total of three rounds. Each round further accumulated a pool of sdAbs binding MIC2. Screening with ELISA showed colonies which panned against EmMIC2, EaMIC2, and/or EtMIC2.
- a fusion protein may comprise an sdAb of the invention bonded directly or through the intermediacy of a linker to another protein or polypeptide.
- the second polypeptide in one embodiment, is not an sdAb and may be any polypeptide.
- a fusion protein comprises two or more sdAbs of the invention, such as 2, 3, 4, 5 or 6 sdAbs, preferably 2, 3, or 4, more typically 2 or 3, more preferably 2.
- the fusion protein may be a homodimer or heterodimer of two or more, such as two sdAbs of the invention.
- a homodimer is defined, in one embodiment, such that the two sdAbs have CDR3 regions which have at least 75% sequence identity to each other.
- the fusion protein may be a homodimer selected from the group consisting of a. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:4 b. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:8; c.
- a first sdAb linked to a second sdAb each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO: 12; d. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO: 16; e. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:20; f.
- a first sdAb linked to a second sdAb each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:24; g. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:28; h. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:32; i. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:36; j.
- a first sdAb linked to a second sdAb each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:40; k. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:44; l. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:48; m.
- each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:52; n. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:56.
- the fusion protein may be a homodimer selected from the group consisting of a. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:20; b. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:24; c. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:28; d.
- a first sdAb linked to a second sdAb each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:36; e. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:40; f. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:48; and g. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:56.
- the fusion protein is a homodimer selected from the group consisting a. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , b. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:5 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:5, c. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:9 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:9, d.
- a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 13 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:13 e. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 17 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:17
- a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 g. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:25 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:25, h.
- a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 29 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:29 i. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:33 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:33
- a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:102 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 102 u. a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:106 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 106, v. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:110 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 110, w. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:114 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 114, x.
- a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:210 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:210 vv. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:214 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:214, ww. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:218 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:218, xx. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:222 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:222, yy.
- the fusion protein is a homodimer selected from the group consisting of a. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 17 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 17, b. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 , c. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:25 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:25, d.
- a single-domain antibody having at least 75% sequence identity to SEQ ID NO:33 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:33 e. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:37 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:37, f. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:45 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:45, and g. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:53 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:53.
- a fusion protein may alternatively be defined, in another embodiment, as a heterodimer comprising two or more sdAbs, such as two sdAbs, of the invention.
- the fusion protein may be a heterodimer selected from the group consisting of a.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:4 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; b.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:8 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; c.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO: 12 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; d.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO: 16 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; e.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:20 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; f.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:24 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; g.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:28 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; h.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:32 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID N0:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:40,
- SEQ ID NO:44 SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; i. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:44 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; l.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:48 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:52 and SEQ ID NO:56; m.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:52 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48 and SEQ ID NO:56; and n.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:56 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48 and SEQ ID NO:52.
- the fusion protein is a heterodimer selected from the group consisting of a. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:20 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; b.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:24 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; c.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:28 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; d.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:36 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; e.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:40 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; f.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:48 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:52 and SEQ ID NO:56 ; and g.
- a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:56 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48 and SEQ ID NO:52.
- fusion proteins that bind EtMIC2 strongly also show binding to EmMIC2. Since EtMIC2 on sequence level is the most divergent of the three MIC2 molecules, this could indicate that fusion proteins binding to EtMIC2 are interacting with a highly conserved binding site. As determined by BLI measurements, all fusion proteins of the invention have demonstrated binding affinities and having and a Kd of less than 10 nM, and less than 1 nM. Of particular interest is the embodiment where the fusion protein is selected from fusion proteins which display cross-binding to both EtMIC2 and EmMIC2, such as SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75.
- the fusion protein is a heterodimer selected from the group consisting of a. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO
- a preferred heterodimer is one in which the fusion protein is selected from the group consisting of a. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 17 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53; b. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53; c.
- the fusion protein of the invention typically comprises two single-domain antibodies covalently bound to each other, preferably at the C-terminus of the first single-domain antibody to the N-terminus of the second single-domain antibody.
- the C-terminus of the first single-domain antibody may be directly covalently bound to the N-terminus of the second single-domain antibody or by means of a linker.
- Preferably the N-terminus of the first single-domain antibody is bound to the C-terminus of the second single-domain antibody by means of a linker.
- the linker is preferably stable to gastric conditions.
- the linker is typically from 1 to 20 nm in length, preferably from 3 to 15 nm, such as 3 to 12 nm.
- the linker is a stable linker and confers stability to the protein of the invention.
- said linker upon exposure as described herein below, said linker is not degraded and hence is capable of maintaining a link between said first and said second sdAb of the present invention.
- the protein is stable in the gastrointestinal tract and/or respiratory system of a subject, i.e., the linker is capable of remaining intact in the gastrointestinal tract and/or respiratory system of a subject.
- the protein is acid stable and/or protease stable.
- the protein may be stable in the presence of any acids, such as bile acid and/or citric acid.
- the fusion protein is also preferably stable, when in a composition or formulation of the invention, in the presence of any proteases, such as pepsin and/or trypsin.
- the fusion protein when in a composition or formulation of the invention, is stable in the presence of gall, such as 50%, 10%, or 2% gall for 1 hour.
- the protein is stable in the presence of a bile salt, such as 0.2 mM, 1 mM, and 10 mM sodium deoxycholate (NaDeox) for 1 hour.
- the protein is temperature stable, such as temperature stable until 70 °C. This indicates high protein stability.
- the protein is pH stable. Unexpectedly, the inventors found that, at pH 3- 4, approx. 70-90% of the protein remained stable, thus said protein is able to pass through the gastrointestinal tract as at least a dimeric fusion protein and retain its activity.
- the first and second scAb are linked via a linker polypeptide comprising 5 to 50 amino acid residues, such as 10 to 40 amino acid residues, such as 10 to 30 amino acid residues.
- the fusion protein comprises two single-domain antibodies covalently bound to each other, wherein the C terminus of the first single-domain antibody is bound to the N terminus of the second single-domain antibody by means of a linker, wherein the linker is a polypetide.
- the linker comprises or consists of glycine and serine residues.
- the linker is a GS linker, such as a GS linker of the structure (GxS)n, where x may be a number between 1 to 10, preferably 3 to 8, such as 2 to 6 or 2 to 5, and n refers to a number of repeats of the GxS sequence, where n may be between 1 to 10, preferably 2 to 5.
- the linker comprises (GGGGS)n where n is 1 to 10, preferably 2 to 5, such as 2, 3, 4 or 5.
- the GS linker is a GGGGS linker, a GGGGSGGGGS linker, a GGGGSGGGGSGGGGS linker, a GGGGSGGGGSGGGGSGGGGS linker, a GGGGSGGGGSGGGGSGGGGSGGGGS linker, or a GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker.
- the linker is (GGGGS)3. The length of the (GGGGS)3 linker was calculated to be about 5.7 nm.
- a further aspect of the invention is directed to a fusion protein selected from the group consisting of: a. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:72; b. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:73; c.
- polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:74; d. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:75; e.
- polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:76; f. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:77; g.
- polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:78; h. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:79; i.
- polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NQ:80; and j. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:81.
- the fusion protein comprises two single-domain antibodies of the invention and has a cross-reactivity by having binding affinity to at least two of EaMIC2, EmMIC2, and EtMIC2 and having a Kd of less than 10 nM to at least one of EaMIC2, EmMIC2, and EtMIC2, such as less than 8 nM, such as less than 7, 6, 5, 4, 3, 2 or 1 nM, preferably a crossreactivity by having a binding affinity to each of EaMIC2, EmMIC2 and EtMIC2.
- the fusion protein of the invention retains binding affinity after thermal treatment such that the fusion protein retains at least 60%, such as at least 65%, such as at least 75% of its binding affinity measured prior to thermal treatment, as performed in Example 7.
- Protein Granules Protein Granules
- the sdAb are typically formulated for use for adminstration to an animal.
- a further aspect of the invention is directed to a composition comprising an sdAb of the invention.
- the invention is directed to a composition comprising the fusion protein of the invention.
- the composition typically further comprises a preservative, filler, and/or carrier material.
- the composition may be a liquid composition, solid composition, solution, dispersion, paste, powder, granule, granulate, coated granulate, tablet, cake, crystal, crystal slurry, gel or pellet.
- the granule suitably comprises:
- the granule comprises:
- the present invention also relates to granules/particles comprising a single-domain antibody of the invention.
- the granule comprises a core, and optionally one or more coatings (outer layers) surrounding the core.
- the core may have a diameter, measured as equivalent spherical diameter (volume based average particle size), of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
- the core diameter, measured as equivalent spherical diameter can be determined using laser diffraction, such as using a Malvern Mastersizer and/or the method described under ISO13320 (2020).
- the core comprises a single-domain antibody or fusion protein of the present invention.
- the core may include additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
- the core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.
- the core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.
- the core may include an inert particle with the single-domain antibody absorbed into it, or applied onto the surface, e.g., by fluid bed coating.
- the core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250- 1200 pm.
- the core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule.
- the optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).
- PEG polyethylene glycol
- MHPC methyl hydroxy-propyl cellulose
- PVA polyvinyl alcohol
- the coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%. The amount may be at most 100%, 70%, 50%, 40% or 30%.
- the coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm.
- the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm.
- the coating should encapsulate the core unit by forming a substantially continuous layer.
- a substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit has few or no uncoated areas.
- the layer or coating should, in particular, be homogeneous in thickness.
- the coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
- fillers e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
- a salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.
- the salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm.
- the salt coating may comprise a single salt or a mixture of two or more salts.
- the salt may be water soluble, in particular, having a solubility at least 0.1 g in 100 g of water at 20 °C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.
- the salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate.
- Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminum.
- Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate.
- alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.
- the salt in the coating may have a constant humidity at 20 °C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate).
- the salt coating may be as described in WO 00/01793 or WO 2006/034710.
- the salt may be in anhydrous form, or it may be a hydrated salt, i.e., a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595.
- anhydrous sodium sulfate Na2SO4
- anhydrous magnesium sulfate MgSO4
- magnesium sulfate heptahydrate MgSO4 ?H2O
- zinc sulfate heptahydrate ZnSO4 ?H2O
- sodium phosphate dibasic heptahydrate Na2HPO4 ?H2O
- magnesium nitrate hexahydrate Mg(NO3)2(6H2O)
- the salt is applied as a solution of the salt, e.g., using a fluid bed.
- the coating materials can be waxy coating materials and film-forming coating materials.
- waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
- PEG poly(ethylene oxide) products
- PEG polyethyleneglycol, PEG
- ethoxylated nonylphenols having from 16 to 50 ethylene oxide units
- ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units
- fatty alcohols fatty acids
- mono- and di- and triglycerides of fatty acids are given in GB 1483591
- the granule may optionally have one or more additional coatings.
- suitable coating materials are polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).
- PEG polyethylene glycol
- MHPC methyl hydroxy-propyl cellulose
- PVA polyvinyl alcohol
- enzyme granules with multiple coatings are described in WO 93/07263 and WO 97/23606.
- the core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
- granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
- Fluid bed granulation involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them to form a granule.
- the cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or enzyme industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90 °C.
- the cores comprising the single-domain antibody contain a low amount of water before coating with the salt. If water sensitive single-domain antibodies are coated with a salt before excessive water is removed, the excessive water will be trapped within the core and may affect the activity of the single-domain antibody negatively.
- the cores preferably contain 0.1-10% w/w water.
- Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and US 4,661 ,452 and may optionally be coated by methods known in the art.
- the granulate may further comprise other actives, such as enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase.
- the one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, betaglucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A
- the present invention also relates to liquid compositions comprising a single-domain antibody of the invention or the fusion protein of the invention.
- the composition may comprise a stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
- a stabilizer include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
- the liquid composition comprising the single-domain antibody or the fusion protein may comprise a stabilizer, e.g., a polyol such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
- the liquid composition may further comprise a filler or carrier material.
- the liquid composition may further comprise a preservative.
- filler(s) or carrier material(s) are included to increase the volume of such compositions.
- suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like.
- Suitable filler or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials.
- the liquid formulation comprises 20-80% w/w of polyol. In one embodiment, the liquid formulation comprises 0.001-2% w/w preservative. In another embodiment, the invention relates to liquid formulations comprising:
- the invention relates to liquid formulations comprising:
- the liquid formulation comprises one or more formulating agents, such as a formulating agent selected from the group consisting of polyol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate.
- a formulating agent selected from the group consisting of polyol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA,
- the polyols is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600, more preferably selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.
- MPG propylene glycol
- the liquid formulation comprises 20-80% polyol (/.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol.
- the liquid formulation comprises 20-80% polyol, e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40%-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600.
- MPG propylene glycol
- the liquid formulation comprises 20-80% polyol (/.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG).
- polyol is selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG).
- the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.
- the liquid formulation comprises 0.02-1.5% w/w preservative, e.g., 0.05-1% w/w preservative or 0.1-0.5% w/w preservative.
- the liquid formulation comprises 0.001-2% w/w preservative (/.e., total amount of preservative), e.g., 0.02- 1.5% w/w preservative, 0.05-1 % w/w preservative, or 0.1 -0.5% w/w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.
- the liquid formulation further comprises one or more enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase.
- the one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, betagalactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha- mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A2, phospholip
- the liquid composition of the invention comprising a single-domain antibody or fusion protein is in the form of an aqueous solution, in particular a solution which is suitable to be added to drinking water for an animal such as a monogastric animal. It is thus contemplated that the single-domain antibodies or fusion proteins may be administered via drinking water rather than in the form of e.g. an animal feed or animal feed additive.
- a further aspect of the invention is directed to animal feed and animal feed additives.
- the invention is directed to a feed additive comprising an sdAb of the invention or the fusion protein of the invention and to a feed comprising said feed additive or an sdAb of the invention or the fusion protein of the invention.
- the present invention relates to animal feed compositions and animal feed additives comprising an sdAb of the invention or a fused protein of the invention, a composition of the invention, a granule of the invention, or a liquid formulation of the invention.
- the feed or feed additive is for a monogastric animal, typically for swine, piglet, growing pig, sow, poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick, cat, dog, or horse.
- the animal feed or animal feed additive is for a monogastric animal from the group consisting of poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick.
- the animal feed or animal feed additive is for a monogastric animal selected from the group consisting of chicken, broiler, layer, and chick.
- the animal feed or animal feed additive is for use in poultry.
- the animal feed or animal feed additive is for a household pet, such as a dog or cat.
- Animal feed compositions or diets have a relatively high content of protein.
- Poultry and pig diets can be characterised as indicated in Table B of WO 01/58275, columns 2-3.
- Fish diets can be characterised as indicated in column 4 of this Table B.
- such fish diets usually have a crude fat content of 200-310 g/kg.
- An animal feed composition according to the invention has a crude protein content of 50- 800 g/kg, and furthermore comprises an sdAb or fused protein of the invention.
- the animal feed composition of the invention has a content of metabolisable energy of 10-30 MJ/kg; and/or a content of calcium of 0.1-200 g/kg; and/or a content of available phosphorus of 0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or a content of methionine plus cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50 g/kg.
- the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO 01/58275 (R. 2-5).
- the nitrogen content is determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC).
- Metabolisable energy can be calculated on the basis of the NRC publication Nutrient requirements in swine, ninth revised edition 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, D.C., pp. 2-6, and the European Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN 90- 71463-12-5.
- the dietary content of calcium, available phosphorus and amino acids in complete animal diets is calculated on the basis of feed tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.
- the animal feed composition of the invention contains at least one vegetable protein.
- the animal feed may comprise vegetable proteins.
- the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w).
- Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae (Leguminosae), Brassicaceae, Amaranthaceae, and Poaceae, such as soybean meal, lupin meal, rapeseed meal, and combinations thereof.
- the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean.
- the vegetable protein source is material from one or more plants of the family Amaranthaceae, e.g. beet, sugar beet, spinach or quinoa.
- Other examples of vegetable protein sources are rapeseed, crambe and cabbage.
- soybean is a preferred vegetable protein source.
- Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.
- the animal feed composition of the invention may also contain animal protein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal, typically in an amount of 0-25%.
- animal feed composition of the invention may also comprise Dried Distillers Grains with Solubles (DDGS), typically in amounts of 0-30%.
- DDGS Dried Distillers Grains with Solubles
- the animal feed composition of the invention may also contain insect protein, such as protein from mealworm, housefly or black soldier fly larvae, typically in meal form. Insect meal may replace fishmeal entirely or in part, and thus may constitute 0-10% of the total feed.
- insect protein such as protein from mealworm, housefly or black soldier fly larvae
- the animal feed composition of the invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or 0-20% whey.
- Animal diets can e.g. be manufactured as mash feed (non-pelleted) or pelleted feed.
- the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question, an sdAb or fused protein of the invention can be added as solid or liquid enzyme formulations.
- an sdAb or fused protein formulation of the invention may be added before or during the ingredient mixing step.
- an sdAb or fused protein preparation may also be added before or during the feed ingredient step.
- an sdAb or fused protein preparation optionally comprises a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets.
- a polyol such as glycerol, ethylene glycol or propylene glycol
- the sdAb or fused protein may also be incorporated in a feed additive or premix.
- the animal feed or animal feed additive comprises one or more enzymes. In an embodiment, the animal feed comprises one or more microbes. In an embodiment, the animal feed comprises one or more vitamins. In an embodiment, the animal feed comprises one or more minerals. In an embodiment, the animal feed comprises one or more amino acids. In an embodiment, the animal feed comprises one or more other feed ingredients.
- the animal feed or animal feed additive comprises the polypeptide of the invention, one or more formulating agents and one or more components selected from the list consisting of: one or more enzymes; one or more microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.
- the animal feed additive comprises one or more formulating agents.
- the animal feed additive comprises one or more probiotics.
- the animal feed additive comprises one or more vitamins.
- the animal feed additive comprises one or more minerals.
- the animal feed additive comprises one or more amino acids.
- the animal feed additive comprises one or more prebiotics.
- the animal feed additive comprises one or more organic acids.
- the animal feed additive comprises one or more phytogenies.
- the invention further relates to a method of improving one or more performance parameters of an animal, comprising administering to an animal the animal feed additive of the invention.
- an animal feed is prepared from the animal feed additive, granule or liquid formulation as described herein and administered to the animal.
- the invention further relates to a method of improving one or more performance parameters of an animal, comprising administering to one or more animals an animal feed or pelleted animal feed comprising the sdAb or fused protein of the invention.
- An aspect of the invention is directed to a method of increasing the food conversion ration (FCR) or body weight gain in animal comprising administering to said animal a feed or feed additive comprising a single-domain antibody or fusion protein of the invention.
- FCR food conversion ration
- ‘improving the performance of an animal’ means that there is an increase in body weight gain. In another embodiment, ‘improving the performance of an animal’ means that there is an improved feed conversion ratio. In a further embodiment, ‘improving the performance of an animal’ means that there is an increased feed efficiency. In a further embodiment, ‘improving the performance of an animal’ means that there is an increase in body weight gain and/or an improved feed conversion ratio and/or an increased feed efficiency.
- the invention in one aspect, is directed to supplementing an animal feed with an sdAb or fused protein of the invention to improve a parameter in an animal, typically for swine, piglet, growing pig, sow, poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick, cat, dog, or horse.
- the parameter is improved in a monogastric animal, such as selected from the group consisting of poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick.
- the parameter is improved in an animal selected from the group consisting of chicken, broiler, layer, and chick.
- the parameter is in a is for a household pet, such as a dog or cat.
- the invention in one aspect, is directed to supplementing an animal feed with an sdAb or fused protein of the invention to improve a parameter in an animal such as a chicken, such as a broiler chicken wherein the parameter is selected from the group consisting of a) growth performance such as weight gain, feed intake, and/or food conversion ratio (FCR); b) macroscopic and histological lesions, c) expression of anti-inflammatory cytokines such as IL10 and IL8; and/or d) carotenoids content in the blood.
- a) growth performance such as weight gain, feed intake, and/or food conversion ratio (FCR)
- FCR food conversion ratio
- b) macroscopic and histological lesions e.g., IL10 and IL8
- carotenoids content in the blood.
- the invention in a further aspect, is directed to supplementing an animal feed with an sdAb or fused protein of the invention to improve a parameter in animal such as a chicken, such as a broiler chicken which is susceptible to infection by Eimeria and Clostridium perfringes, wherein the parameter is selected from the group consisting of a) growth performance such as weight gain, feed intake, and/or food conversion ratio (FCR); b) macroscopic and histological lesions, c) expression of anti-inflammatory cytokines such as IL10 and IL8; and/or d) carotenoids content in the blood.
- a) growth performance such as weight gain, feed intake, and/or food conversion ratio (FCR)
- FCR food conversion ratio
- b) macroscopic and histological lesions c
- expression of anti-inflammatory cytokines such as IL10 and IL8
- carotenoids content in the blood.
- the invention is furthermore directed to a method of treating, preventing or improving infections, such as Eimeria and Clostridium perfringens infections, or other parasitic infections, in a monogastric animal comprising administering to the animal a composition, an animal feed or an animal feed additive comprising an sdAb or fused protein of the invention.
- infections such as Eimeria and Clostridium perfringens infections, or other parasitic infections
- one embodiment of the invention relates to sdAbs or fusion proteins of the invention which limit mobility of the parasite and/or reduces invasion of intestinal cells. If the parasite lifecycle is inhibited, the risk of coccidiosis is lowered. Accordingly, one embodiment of the invention relates to sdAbs or fusion proteins of the invention which limit mobility of the parasite and/or reduces invasion of intestinal cells and/or inhibits the lifecycle of the parasite.
- one embodiment of the invention relates to sdAbs or fusion proteins of the invention which limit mobility of any one of Eimeria tenella, Eimeria maxima, and Eimeria acervulina and/or reduces invasion of intestinal cells and/or inhibits the lifecycle of any one of Eimeria tenella, Eimeria maxima, and Eimeria acervulina.
- ‘Microneme proteins’ are essential for host invasion and movement.
- a further aspect of the invention is directed to to sdAbs or fusion proteins of the invention which binding to Microneme proteins, preferably Microneme protein 2 (MIC2).
- the parasitic infection is typically in an animal selected from the group consisting of swine, piglet, growing pig, sow, poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick, cat, dog, horse.
- the parasitic infection is in an animal selected from the group consisting of poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick.
- the parasitic infection is typcially in an animal selected from the group consisting of chicken, broiler, layer, and chick.
- the parameter is in a is for a household pet, such as a dog or cat.
- An aspect of the invention is directed supplementing an animal feed with a single-domain antibody or fusion protein of the invention, to treat, prevent or improve/reduce an infection, such as Eimeria and Clostridium perfringens infections, in a monogastric animal, compared to an animal feed without the supplementation.
- a further aspect of the invention is directed to a method of treating or preventing coccidiosis in poultry comprising the use of a granule, composition, feed additive or feed of the invention.
- the invention is directed to a use of a fusion protein or an sdAb for the preparation of a medicament for the prevention or treatment of coccidiosis in poultry.
- a further aspect is directed to a method of preventing or treating Eimeria or coccidiosis in poultry comprising administering a feed or additive of the invention.
- the invention is furthermore directed to a method of reducing parasites or reducing the risk of infections in an animal, such as an animal selected from the group consisting of swine, piglet, growing pig, sow, poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick, and horse.
- a further embodiment is directed to a method of reducing parasites or reducing the risk of infections in poultry.
- a further aspect is directed to a method of reducing parasites or reducing the risk of infections in a pet, such as but not limited to a dog, cat, bird, or rabbit.
- the sdAb or fused protein of the invention may be fed to the animal from birth until slaughter.
- the sdAb or fusion protein of the invention is fed to the animal on a daily basis from birth until slaughter. More preferably, the sdAb or fusion protein of the invention is fed to the animal on a daily basis for at least 10 days, such as at least 15 days or at least 20 days (where the days can be continuous or non-continuous) during the life span of the animal.
- the sdAb or fusion protein of the invention is fed to the animal for 10-20 days followed by a non-treatment period of 5-10 days, and this cycle is repeated during the life span of the animal.
- the sdAb or fusion protein of the invention may be fed to broilers for the first 49 days after hatching.
- the sdAb or fusion protein of the invention is fed to broilers for the first 36 days after hatching. More preferably, the sdAb or fusion protein of the invention is fed to broilers on days 22 to 36 after hatching. Further preferably, the sdAb or fusion protein of the invention is fed to broilers during the pre-starter (days 1-7) period. Further preferably, the sdAb or fusion protein of the invention is fed to broilers during the starter (days 8-22) period.
- the sdAb or fusion protein of the invention is fed to broilers during the pre-starter (days 1- 7) and starter (days 8-22) period.
- the sdAb or fusion protein of the invention may be fed to layers during the life span of the animal.
- the sdAb or fusion protein of the invention is fed to layers for 76 weeks from hatching. More preferably, the sdAb or fusion protein of the invention is fed to layers during the laying period, (from ca. week 18). Further preferably, the sdAb or fusion protein of the invention is fed to layers during the laying period but withheld during the forced molting period.
- the sdAb or fusion protein of the invention may be fed to turkeys during life span of the animal.
- the sdAb or fusion protein of the invention is fed to turkeys for 24 weeks from hatching. More 30 preferably, the sdAb or fusion protein of the invention is fed to turkeys for the first 16 weeks from hatching (for hens) and for the first 20 weeks for hatching (for toms).
- the sdAb or fusion protein of the invention may be fed to swine during life span of the animal.
- the sdAb or fusion protein of the invention is fed to swine for 27 weeks from birth.
- the sdAb or fusion protein of the invention is fed to piglets from birth to weaning (at 4 weeks). Further preferably, the sdAb or fusion protein of the invention is fed to piglets for the first 6 weeks from birth (4 weeks of lactation and 2 weeks post-weaning). Further preferably, the sdAb or fusion protein of the invention is fed to weaning piglets during the pre-starter (days 1-14 after weaning) Further preferably, the sdAb or fusion protein of the invention is fed to weaning piglets during the starter (days 15-42 after weaning) period.
- the sdAb or fusion protein of the invention is fed to weaning piglets during the pre-starter (days 1-14 after weaning) and starter (days 15-42 after weaning) period. Further preferably, the sdAb or fusion protein of the invention is fed to swine during the grower/fattening period (week 10 to ca. week 27 after birth).
- a further aspect of the invention is directed to an sdAb or fusion protein comprising an sdAb having a binding affinity for the antigen microneme protein 2 as defined by the invention for the preparation of an animal feed additive or animal feed.
- the sdAb of the invention or the fusion protein of the invention may also be formulated as animal feed or animal feed additive for treating, preventing, or reducing the spread of Eimeria or Clostridium perfringens infections, of an animal.
- the single-domain antibodies or fusion proteins may be administered not only as part of an animal feed or animal feed additive, but alternatively or additionally via drinking water.
- One aspect of the invention thus relates to method for treating, preventing, or reducing the spread of Eimeria or Clostridium perfringens infections in an animal such as a monogastric animal, the method comprising administering to the animal drinking water comprising a single-domain antibody or fusion protein of the invention.
- One aspect of the invention is directed to use of a composition, an animal feed, or an animal feed additive for treating, preventing, or reducing the spread of Eimeria and Clostridium perfringens infections of a monogastric animal wherein the composition, the animal feed or the animal feed additive comprises the fusion protein or sdAb of the invention.
- the invention is directed to a method of treating, preventing or or reducing the spread of Eimeria and Clostridium perfringens infections, of a monogastric animal comprising administering to the animal a composition, an animal feed or an animal feed additive comprising the fusion protein or sdAb of the invention.
- the animal is a bird, more typically poultry.
- An aspect of the invention is directed to use of an sdAb or fusion protein comprising an sdAb having a binding affinity for the antigen microneme protein 2 (MIC2) in an animal feed or animal feed additive for the prevention or treatment of a parasitic infection, wherein the sdAb is selected from the group consisting of:
- (bf) a single-domain antibody derived from the single-domain antibody of (a), (b), (c), (d), (e), (f). (g). (h), (i), 0), (k), (I), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), (x), (y), (z), (aa), (ab), (ac), (ad), (ae), (af), (ag), (ah), (ai), (aj), (ak), (al), (am), (an), (ao), (ap), (aq), (ar), (as), (at), (au), (av), (aw), (ax), (ay), (az), (ba), (bb) or (be), wherein the N- and/or C-terminal end has been extended by addition of one or more amino acids.
- this aspect of the invention is directed to a single-domain antibody having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53.
- a further aspect of the invention is directed to use of an sdAb or fusion protein comprising an sdAb having a binding affinity for the antigen microneme protein 2 (MIC2) in an animal feed or animal feed additive wherein the sdAb is selected from the group consisting of wherein the single-domain antibody comprises a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44,
- SEQ ID NO:62 SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,
- SEQ ID NO:68 SEQ ID NO:69, SEQ ID NQ:70 and SEQ ID NO:71.
- An aspect of the invention is directed to use of a fusion protein having a binding affinity for the antigen microneme protein 2 (MIC2) in an animal feed or animal feed additive for the prevention or treatment of a parasitic infection, wherein the fusion protein selected from the group consisting of: a. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:72; b.
- polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:73; c. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:74; d.
- polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:75; e. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:76; f.
- polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:77; g. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:78; h.
- polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:79; i. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NQ:80; and j.
- polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:81.
- a further aspect of the invention is directed to a method of reducing the likelihood the spread of Eimeria in animals comprising the use of an sdAb, fusion protein, granule, composition, feed additive, or feed of the invention.
- a further aspect of the invention is directed to an sdAb or fusion protein comprising an sdAb having a binding affinity for the antigen microneme protein 2 as defined by the invention for the preparation of an animal feed additive or animal feed.
- One aspect of the invention is directed to use of a composition, an animal feed, or an animal feed additive for treating, preventing or or reducing the spread of Eimeria and Clostridium perfringens infections of a monogastric animal wherein the composition, the animal feed or the animal feed additive comprises the fusion protein or sdAb of the invention.
- the invention is directed to a method of treating, preventing or or reducing the spread of Eimeria and Clostridium perfringens infections, of a monogastric animal comprising administering to the animal a composition, an animal feed or an animal feed additive comprising the fusion protein or sdAb of the invention.
- the animal is a bird, more typically poultry.
- the single-domain antibodies of the invention, or fusion proteins comprising multimer components of the single-domain antibodies, as well as compositions comprising the single-domain antibodies or fusion proteins, and methods using the single-domain antibodies or fusion proteins are capable of preventing or reducing infection levels of Eimeria or Clostridium perfringens infections in a monogastric animal by at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, compared to infection levels without the treatment by the single-domain antibodies or fusion proteins.
- a further aspect is directed to method of reducing the levels coccidiocidal or coccidiostatic agents in animal feed or animal feed additives comprising the use of an sdAb or fuion protein of the invention.
- This may be a method of lowering the amounts of one or more agents selected from ionophores, such as Maduramicin, Salinomycin, Lasalocid, Monensin, Narasin, Semduramicin; Amprolium, Nicarbazin; Sulfonamides such as Sulphadimidine, Sulphaquinoxaline, Sulphadimethoxine, Sulphanitran and Sulphaguanidine: Ethopabate; Quinolones such as Burquinolate, Decoquinate, Clopidol, Robenidine, Halofuginone and Diclazuri in animal feed comprising the use of use of an sdAb or fusion protein of the invention.
- ionophores such as Maduramicin, Salinomycin, Lasalocid,
- the fusion proteins of the invention are a solution to significantly reduce antimicrobial resistance (AMR). Extensive and arguably unnecessary use of antimicrobial agents has led to AMR development in different pathogenic microorganisms.
- AMR antimicrobial resistance
- one aspect of the invention is directed to a method to control, prevent, and treat infections caused by the Eimeria parasite, particularly in the poultry industry.
- the poultry industry is in serious need of new techniques to control, prevent, and treat infections caused by parasites.
- the fusion proteins of the invention can reduce the need for anticoccidial agents in the poultry industry.
- the fusion proteins of the invention inactivate virulence factors by blocking their binding site, which means the virulence factor is unable to bind.
- a virulence factor is a protein of a pathogenic bacteria, virus, fungi, or parasite that initiates or facilitates infection. Without virulence factors, the pathogen would not be harmful since it is not able to cause infection.
- the mode of action of fusion proteins involves their role as virulence inactivating proteins.
- the fusion proteins of the invention are proteins which strengthen the gut microbiome and reduce the risk of gastrointestinal infections.
- the fusion proteins of the invention bind extracellular virulence factors in the gastrointestinal tract so per se they do not directly kill or inhibit growth of pathogens.
- the fusion protein binding inactivates virulence factors, and as a result, the pathogen is not able to establish a gastrointestinal infection and symptoms are minimized.
- the fusion protein is designed to bind to and block a particular binding site, active site, or receptor of a specific virulence factor from a pathogen.
- the desired binding site to be blocked is highly conserved, and if the pathogen mutates, it will lose its binding affinity and is not able to interact with or infect the host anymore.
- the mutation is thus a loss of function mutation. If the microorganism tries to escape the blocking of its virulence factor by mutating the specific binding site blocked, the virulence factor will lose function as it is not be able to bind to the binding site of the host cell.
- a benefit from the use of fusion protein of the invention is a reduced use of antimicrobial agents, and the occurrence and spreading of antimicrobial resistance will decrease because the selective pressure on microorganisms is reduced.
- the invention fusion proteins of the invention are used to reduce antimicrobial resistance in animals.
- the virulence factors of pathogens initiate infection in the host.
- Pathogens have different types of virulence factors: toxins that are excreted, and adhesins that are surface bound.
- the different types of virulence factors in different pathogens initiate and accelerate infection in the host.
- a pathogen with inactivated virulence factors can live in a host without infecting the host.
- the mode of action of fusion proteins is to block binding sites of virulence factors, thereby inactivating virulence, without interacting intracellularly with the pathogen.
- the fusion proteins of the invention physically block the binding site of the virulence factor, disabling the virulence factor from binding to the host cell receptor, thereby preventing the pathogen from establishing an infection as intended.
- the selective pressure that antimicrobial agents induce is not the same stress as fusion proteins induce.
- the difference is that the mode of action of antimicrobial agents is to kill the microorganisms or inhibit the growth of them intracellularly.
- the microorganisms are attacked and only mutants with resistance surivive.
- Resistant variants are selected for and will expand through clonal expansion.
- the mode of action of fusion proteins is to block the binding site of the virulence factor of the microorganism with high affinity extracellularly. The microorganisms are not attacked, and resistant variants will not be selected for.
- a further aspect of the invention is directed to a method of reducing antimicrobial resistance in animals, such as in poultry farming, suitably comprising a method of reducing antimicrobial resistance in an animal population comprising blocking binding sites of virulence factors, thereby disabling the virulence factor from binding to the host cell receptor, thereby preventing the pathogen from establishing an infection, said method comprising administering to said animal a feed or feed additive comprising a single-domain antibody or fusion protein of the invention.
- the present invention also relates to methods of producing a single-domain antibody of the present invention, comprising (a) cultivating a cell, under conditions conducive for production of the single-domain antibody; and optionally, (b) recovering the single-domain antibody.
- a method of production of the fusion proteins comprises the use of a polynucleotide encoding a signal peptide which is operably linked to a polynucleotide encoding a fusion protein selected from the group consisting of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80 and SEQ ID NO:81 , which is heterologous to the signal peptide.
- a method of production of single-domain antibodies comprises the use of a polynucleotide encoding a signal peptide which is operably linked to a polynucleotide encoding a single-domain antibody selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126
- An aspect of the invention is directed to a polynucleotide encoding a propeptide operably linked to the polynucleotide of the invention, encoding a single-domain antibody which is heterologous to the propeptide.
- the invention is further directed to a nucleic acid construct or expression vector comprising the polynucleotide encoding a fusion protein selected from the group consisting of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80 and SEQ ID N0:81.
- the nucleic acid construct or expression vector may comprise a gene encoding a protein operably linked to the polynucleotide wherein the gene is heterologous to the polynucleotide encoding the propeptide.
- the invention is further directed to a nucleic acid construct or expression vector comprising a polynucleotide encoding a single-domain antibody selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ
- the nucleic acid construct or expression vector may comprise a gene encoding a protein operably linked to a gene encoding a protein operably linked to a protein operably linked to a protein operably linked to a protein operably linked to a protein operably linked to a protein operably linked to a protein operably linked to a protein operably linked to a protein operably linked to a protein operably linked to a protein operably linked to a protein operably a protein sequence of the sequence.
- SEQ ID NO:158 SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, S
- a further aspect is directed to a recombinant host cell comprising a nucleic acid construct or expression vector of the invention.
- the invention is directed to a method of producing a singledomain antibody or fusion protein of the invention, comprising cultivating the recombinant host cell comprising a nucleic acid construct or expression vector of the invention under conditions conducive for production of the protein.
- the method typically further comprises recovering the protein.
- the invention is furthermore directed to a recombinant host cell comprising a nucleic acid construct or expression vector of the invention and to a method of producing a protein, comprising cultivating the recombinant host cell under conditions conducive for production of the protein.
- the method typically further comprises recovering the protein.
- the present invention also relates to methods of producing a single-domain antibody of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the single-domain antibody; and optionally, (b) recovering the single-domain antibody.
- the host cell is cultivated in a nutrient medium suitable for production of the single-domain antibody using methods known in the art.
- the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state, and/or microcarrier-based fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the single-domain antibody to be expressed and/or isolated.
- suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the single-domain antibody is secreted into the nutrient medium, the single-domain antibody can be recovered directly from the medium. If the single-domain antibody is not secreted, it can be recovered from cell lysates.
- the single-domain antibody may be detected using methods known in the art that are specific for the antibodys, including, but not limited to, the use of specific antigens, DELFIA and ELISA.
- the single-domain antibody may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
- a whole fermentation broth comprising the singledomain antibody is recovered.
- a cell-free fermentation broth comprising the single-domain antibody is recovered.
- the single-domain antibody may be purified by a variety of procedures known in the art to obtain substantially pure single-domain antibodies and/or single-domain antibody fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science-, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10). In an alternative aspect, the single-domain antibody is not recovered.
- the polynucleotide may also be mutated by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the single-domain antibody, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence.
- nucleotide substitutions see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107.
- the polynucleotide is isolated.
- the polynucleotide is purified.
- the present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
- the polynucleotide may be manipulated in a variety of ways to provide for expression of the single-domain antibody. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
- the control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a single-domain antibody of the present invention.
- the promoter contains transcriptional control sequences that mediate the expression of the single-domain antibody.
- the promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular single-domain antibodies either homologous or heterologous to the host cell.
- Suitable promoters for directing transcription of the polynucleotide of the present invention in a bacterial host cell are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY, Davis et al., 2012, supra, and Song et a!., 2016, PLOS One 11(7): e0158447.
- promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor and Trichoderma cells, such as the promoters described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
- the control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription.
- the terminator is operably linked to the 3’-terminus of the polynucleotide encoding the single-domain antibody. Any terminator that is functional in the host cell may be used in the present invention.
- Preferred terminators for bacterial host cells may be obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
- aprH Bacillus clausii alkaline protease
- AmyL Bacillus licheniformis alpha-amylase
- rrnB Escherichia coli ribosomal RNA
- Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
- Preferred terminators for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.
- Other useful terminators for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
- control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
- mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacterid. 177: 3465-3471).
- mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Ce// 5(11): 1838-1846.
- the control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell.
- the leader is operably linked to the 5’-terminus of the polynucleotide encoding the single-domain antibody. Any leader that is functional in the host cell may be used.
- Suitable leaders for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12): 3051-3059, and by Kaberdin and Blasi, 2006, FEMS Microbiol. Rev. 30(6): 967-979.
- Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
- Suitable leaders for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
- ENO-1 Saccharomyces cerevisiae enolase
- Saccharomyces cerevisiae 3-phosphoglycerate kinase Saccharomyces cerevisiae alpha-factor
- Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase ADH2/GAP
- the control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the polynucleotide which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
- Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
- Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.
- the control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a single-domain antibody and directs the single-domain antibody into the cell’s secretory pathway.
- the 5’-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the singledomain antibody.
- the 5’-end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence.
- a heterologous signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence.
- a heterologous signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance secretion of the singledomain antibody. Any signal peptide coding sequence that directs the expressed single-domain antibody into the secretory pathway of a host cell may be used.
- Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alphaamylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Freudl, 2018, Microbial Cell Factories 17: 52.
- Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptide described by Xu et al., 2018, Biotechnology Letters 40: 949-955
- Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.
- the control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a single-domain antibody.
- the resultant single-domain antibody is known as a proenzyme or prosingle-domain antibody (or a zymogen in some cases).
- a prosingle-domain antibody is generally inactive and can be converted to an active singledomain antibody by catalytic or autocatalytic cleavage of the propeptide from the prosingledomain antibody.
- the propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
- the propeptide sequence is positioned next to the N-terminus of a single-domain antibody and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
- the single-domain antibody may comprise only a part of the signal peptide sequence and/or only a part of the propeptide sequence.
- the final or isolated single-domain antibody may comprise a mixture of mature single-domain antibodies and single-domain antibodies which comprise, either partly or in full length, a propeptide sequence and/or a signal peptide sequence.
- regulatory sequences that regulate expression of the single-domain antibody relative to the growth of the host cell.
- regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
- Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems.
- yeast the ADH2 system or GAL1 system may be used.
- the Aspergillus niger glucoamylase promoter In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used.
- Other examples of regulatory sequences are those that allow for gene amplification. In fungal systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals.
- the control sequence may also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding single-domain antibody that controls the rate of the transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence.
- the transcription factor may function alone and/or together with one or more other single-domain antibodies or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase.
- Transcription factors are characterized by comprising at least one DNA-binding domain which often attaches to a specific DNA sequence adjacent to the genetic elements which are regulated by the transcription factor.
- the transcription factor may regulate the expression of a protein of interest either directly, i.e., by activating the transcription of the gene encoding the protein of interest by binding to its promoter, or indirectly, i.e., by activating the transcription of a further transcription factor which regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the further transcription factor.
- Suitable transcription factors for fungal host cells are described in WO 2017/144177.
- Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011 , Subcellular Biochemistry 52: 7-23, as well in Balleza et al., 2009, FEMS Microbiol. Rev. 33(1): 133-151.
- the present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals.
- the various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the single-domain antibody at such sites.
- the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression.
- the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
- the recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide.
- the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
- the vector may be a linear or closed circular plasmid.
- the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
- the vector may contain any means for assuring self-replication.
- the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
- a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
- the vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells.
- a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
- the vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
- the vector may rely on the polynucleotide’s sequence encoding the single-domain antibody or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ).
- homologous recombination such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ).
- the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
- the origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell.
- the term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo. More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a single-domain antibody. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell.
- An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
- the present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a single-domain antibody of the present invention.
- a construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier.
- the choice of a host cell will to a large extent depend upon the gene encoding the single-domain antibody and its source.
- the single-domain antibody can be native or heterologous to the recombinant host cell.
- at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the single-domain antibody.
- the recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotide of the present invention.
- the host cell may be any microbial cell useful in the recombinant production of a singledomain antibody of the present invention, e.g., a prokaryotic cell or a fungal cell.
- the prokaryotic host cell may be any Gram-positive or Gram-negative bacterium.
- Grampositive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.
- Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.
- the prokaryotic host cell is E.coli.
- the bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
- the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus subtilis cell.
- Bacillus classes/genera/species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70: 406-438.
- the bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.
- the bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.
- Methods for introducing DNA into prokaryotic host cells are well-known in the art, and any suitable method can be used including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, with DNA introduced as linearized or as circular polynucleotide. Persons skilled in the art will be readily able to identify a suitable method for introducing DNA into a given prokaryotic cell depending, e.g., on the genus. Methods for introducing DNA into prokaryotic host cells are for example described in Heinze et al., 2018, BMC Microbiology 18:56, Burke et al., 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294, Choi et al., 2006, J. Microbiol. Methods Q4: 391-397, and Donald et al., 2013, J. Bacteriol. 195(11): 2612- 2620.
- the host cell may be a fungal cell.
- “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
- Fungal cells may be transformed by a process involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation as reviewed by Li et al., 2017, Microbial Cell Factories 16: 168 and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, Christensen etal., 1988, Bio/TechnologyQ: 1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27: 53-75.
- any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as linearized or as circular polynucleotide.
- the yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
- the yeast host cell is a Pichia or Komagataella cell, e.g., a Pichia pastoris cell (Komagataella phaffii).
- the host cell is isolated. In another aspect, the host cell is purified.
- a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56.
- the single-domain antibody according to embodiment 1 comprising a CDR3 region having at least 80% sequence identity, such as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96, 97, 98, or 99% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 SEQ ID NO:56.
- the single-domain antibody according to embodiment 1 comprising a CDR3 region a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56, such as a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, and SEQ ID NO:56, typically a sequence selected from the group consisting of SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:16
- a single-domain antibody having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53.
- the single-domain antibody of embodiment 8, comprising, consisting essentially of, or consisting of a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, preferably SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, more preferably SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO
- a single-domain antibody having a binding affinity for the antigen microneme protein 2 such as a binding affinity for one or more of MIC2 from Eimeria tenella (EtMIC2), MIC2 from Eimeria maxima (EmMIC2), and MIC2 from Eimeria acervulina EaMIC2.
- the single-domain antibody according to embodiment 10 or 11 having a Kd for EmMIC2 of 10 nM or less, such as of 8 nM or less, such as 7, 6, 5, 4 3, 2, or 1 nM or less.
- the single-domain antibody according to embodiment 14 having a Kd for EmMIC2 of less than 10 nM wherein the CDR3 region has at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 57 and SEQ ID NO: 58 of Family 8 and SEQ ID NO:20, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO: 64 of Family 21 .
- the single-domain antibody according to embodiment 16 having cross-reactivity by binding to at least two of EaMIC2, EmMIC2 and EtMIC2 and comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71 of Family 23.
- the single-domain antibody according to any of embodiments 10 to 19, having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, preferably SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, more preferably SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID
- fusion protein according to embodiment 21 comprising a homodimer or heterodimer of two sdAb as defined by any of embodiments 1-20.
- a fusion protein selected from the group consisting of: a. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:72; b. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:73; c.
- a polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:74; d. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:75; e.
- polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:76; f. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:77; g.
- a polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:78; h. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:79; i.
- polypeptide having at least 75% sequence identity such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NQ:80; and j. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:81.
- the fusion protein according to any of embodiments 21 to 25 having a binding affinity of 10 nM or less, such as of 8 nM or less, such as 7, 6, 5, 4 3, 2, or 1 nM or less
- the fusion protein according to any of embodiments 21 to 26 having a retained binding affinity after thermal treatment under method A, of at least 60%, such as at least 65%, such as at least 75%.
- composition comprising an sdAb as defined by any of embodiments 1 to 20.
- composition comprising the fusion protein as defined by any of embodiments 21 to 27.
- a granule which comprises:
- a granule which comprises:
- a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises the single-domain antibody of any one of embodiments 1 to 20 or the fusion protein of any of embodiments 21 to 27.
- a liquid composition comprising the single-domain antibody of any one of embodiments 1 to 20 or the fusion protein of any of embodiments 21 to 27, and a stabilizer, e.g., a polyol such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
- a stabilizer e.g., a polyol such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
- liquid composition of embodiment 33 further comprising a filler or carrier material.
- composition comprising the single-domain antibody of any one of embodiments 1 to 20 or the fusion protein of any of embodiments 21 to 27, the granule of embodiments 31 or 32, or the liquid composition of any one of embodiments 33 to 35.
- composition of embodiment 36 which is a liquid composition, solid composition, solution, dispersion, paste, powder, granule, granulate, coated granulate, tablet, cake, crystal, crystal slurry, gel or pellet.
- a feed additive comprising an sdAb as defined by any of embodiments 1 to 20 or the fusion protein as defined in any of embodiments 21 to 27.
- a feed comprising a feed additive as defined by embodiment 39.
- a method of reducing the likelihood of the spread of Eimeria comprising the use of a granule, composition, feed additive, or feed defined by any of embodiments 28 to 40
- a method of reducing antimicrobial resistance in an animal population comprising blocking binding sites of virulence factors, thereby disabling the virulence factor from binding to the host cell receptor, thereby preventing the pathogen from establishing an infection, said method comprising administering to said animal a feed or feed additive comprising a singledomain antibody as defined in any of embodiments 1 to 20 or fusion protein as defined by any of embodiments 21 to 27.
- a method of increasing the feed conversion ration (FCR) or body weight gain in animals comprising administering to said animal a feed or feed additive comprising a single-domain antibody as defined in any of embodiments 1 to 20 or fusion protein as defined by any of embodiments 21 to 27.
- a method of treating or preventing coccidiosis in poultry comprising the use of a granule, composition, feed additive, or feed defined by any of embodiments 28 to 40
- a method of reducing parasites or reducing the risk of infections in a pet such as but not limited to a dog, cat, bird or rabbit.
- a nucleic acid construct or expression vector comprising the polynucleotide of embodiment 47, operably linked to one or more control sequences that direct the production of the single-domain antibody in an expression host.
- a recombinant host cell comprising the nucleic acid construct or expression vector of embodiment 48.
- a method of producing a single-domain antibody having binding activity comprising cultivating the recombinant host cell of embodiment 49 under conditions conducive for production of the single-domain antibody.
- a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NQ:60, SEQ ID N0:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70 and SEQ ID N0:71.
- the single-domain antibody according to embodiment 51 further comprising a CDR1 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NQ:30, SEQ ID NO:42, SEQ ID NQ:50, SEQ ID NO:54, SEQ ID NQ:107, SEQ ID NO:111 , SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:147, SEQ ID NO:159, SEQ ID NO:163, SEQ ID NO:183, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:199, SEQ ID NO:211 , SEQ ID NO:215 and SEQ ID NO:239 SEQ ID NO:2, SEQ ID NO:6, SEQ ID NQ:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:
- the single-domain antibody according to embodiment 51 or 52 further comprising a CDR2 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:39, SEQ ID NO:51 , SEQ ID NO:136, SEQ ID NO:164, SEQ ID NO:172, SEQ ID NQ:180, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:192, SEQ ID NO:212 and SEQ ID NO:236 SEQ ID NO:3, SEQ ID NO:7, SEQ ID NQ:10, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID
- a fusion protein comprising two or more sdAbs as defined by any of embodiments 51-54.
- a fusion protein as defined by any of embodiments 21-27 or 54, or an sdAb as defined by any of embodiments 1-20 or 51-53, for use as a medicament.
- the fusion protein of embodiment 56 for use in the prevention or treatment of coccidiosis in poultry.
- An aqueous suitable for adding to drinking water for an animal such as a monogastric animal wherein the aqueous solution comprises a single-domain antibody or fusion protein as defined in any of the previous embodiments.
- a method for treating, preventing, or reducing the spread of Eimeria or Clostridium perfringens infections in an animal such as a monogastric animal comprising administering to the animal drinking water comprising a single-domain antibody or fusion protein as defined in any of the previous embodiments.
- DELFIA time-resolved fluorescence normalized assay
- biotinylated antigen (MIC2) was added at 50 nM in 3% milk-PBS and incubated for 1 h at room temperature.
- streptavidin-conjugated europium (Perkin Elmer, #1244-360) was added dilute 1/500 in DELFIA assay buffer (Perkin Elmer #1244-111) and incubated for 30 minutes at room temperature.
- europium fluorescence was activated using DELFIA enhancement solution (Perkin Elmer #4001-0010). Fluorescence intensity was determined using a microplate reader measuring emission at 615 nm.
- proteins were kept in a steam box followed by quantification of binding via DELFIA as described above.
- proteins were subjected to 5 ll/ml pepsin at pH 3.0 for 15 min followed by quantification of binding via DELFIA as described above.
- the genes encoding the desired products were synthesized as linear DNA fragments with appropriate cloning sites in the 3’ and 5’ ends. These were then sub-cloned into the multiple cloning site of a standard expression vector with Lacl induced expression and kanamycin resistance gene selection. For cloning, restriction cloning was applied with Notl and Ncol used as restriction enzymes. The plasmid was subsequently transformed into an E. coli expression strain and streaked to single colonies. A single colony was picked, outgrown in LB with kanamycin (50 pg/ml), and finally stored as single use glycerol stock aliquots. To avoid destructive mutations, gene inserts were sequenced for each expression construct.
- kanamycin 50 pg/ml was inoculated with glycerol stock and incubated overnight at 37 °C and 250 rpm. The following day overnight culture was transferred to autoinduction media and incubated for 2 hrs at 37 °C and 250 rpm followed by another 22 hrs at 25 °C and 250 rpm. The culture was then harvested via centrifugation at 5.000 g for 15 min. The resulting E. coli biomass was stored at -20 °C.
- E. coli biomass containing target protein was thawed and suspended in ice-cold TES buffer (30 mM Tris-HCI, pH 8.0, 1 mM EDTA, 20% Sucrose, 1xPBS) with protein inhibitor cocktail tablet, lysozyme, and benzonase. After 20 min on ice, the suspension was spun down at 15.000 g for 15 minutes. The supernatant was retained and kept on ice. The pellet was then resuspended in ice-cold 5 mM MgSO4 with protein inhibitor cocktail tablet, lysozyme, and benzonase. The suspension was incubated on ice for 20 minutes, before it was spun down at 15.000 g for 15 minutes. The supernatant was pooled with the retained supernatant and spun down at 21.000 g for 60 minutes. The resulting final supernatant was collected.
- TES buffer 30 mM Tris-HCI, pH 8.0, 1 mM EDTA, 20% Suc
- Nickel affinity columns for immobilized metal affinity chromatography (IMAC) in a gravitybased setup were used for first step of purification.
- the column was equilibrated with wash buffer (1xPBS with 200 mM NaCI and 20 mM imidazole, pH 8.0) followed by loading the final supernatant. After the supernatant, wash buffer was used to wash the column, before eluting with elution buffer (1xPBS with 200 mM NaCI and 500 mM imidazole, pH 8.0). The protein was then dialyzed overnight into 1xPBS.
- KD values were guantified via bio-layer interferometry (BLI).
- BLI bio-layer interferometry
- BLI is dependent on the size of the molecules measured. The smaller the molecule, the lower the signal intensity.
- measurements are performed at sdAb concentrations above and below the global KD value. However, also here, the lower the sdAb concentration, the lower the signal. In effect, this creates a lower threshold for KD determination for sdAbs. For sdAbs, this threshold for KD measurement is around 0.5-1 .0 nM.
- the sdAbs for which it is was not possible to determine KD were also generally the sdAbs displaying highest TRF signal in Figure 3. This validates the correlation between TRF signal and binding affinity.
- Fusion protein expression constructs were designed as homodimers from sdAbs as presented above.
- a (GGGGS)3-linker was used to link the two sdAb seguences.
- Fusion proteins were expressed using a standard expression vector, encoding C-terminal 6xHis-tag and FLAG-tag as well as an N-terminal signal peptide for transfer to the periplasm of E. coli BL21 . As with antigens and sdAbs, also fusion proteins were purified using IMAC and I EX.
- Fusion proteins were expressed in the periplasm of an E. coli BL21 strain using standard protein expression methods. These were subseguently purified using IMAC followed by I EX. Fusion proteins were expressed and purified to a minimum of 90% purity.
- fusion proteins which showed affinity, also displayed significantly higher affinity for their target antigen compared to the corresponding monovalent sdAbs.
- KD values were guantified using BLI.
- KD values for the fusion proteins of the invention have been demonstrated to be lower than 1 nM.
- a desired trait of fusion protein is cross-reactivity, meaning the ability of a fusion protein to bind other MIC2 variants than the one it was panned against. To test for cross-reactivity, a standard DELFIA assay was applied.
- fusion proteins of the invention have binding affinities with a Kd of less than 1 nM, the success criterion.
- the fusion protein is selected from fusion proteins which display cross-binding to both EtMIC2 and EmMIC2 such as SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75.
- Fecal material was sampled from 10 different chicken houses. Eimeria spp. oocysts were extracted based on established methods and homogenized in a compact bead mill using glass beads to break the oocyst membrane, releasing the individual sporozoites. Homogenized oocysts were then mixed in equal ratios to represent the broadest possible selection of industrially occurring parasite load. For comparison, a commercially available vaccine (Evalon, HIPRA) comprising attenuated Eimeria spp. oocysts was used. In this vaccine oocysts of five Eimeria spp. species are present.
- Fusion proteins showed binding to the oocyst material from both fecal matter and vaccine solution. This very strong in vitro validation for fusion proteins to also bind live Eimeria spp. parasites in vivo was surprising.
- Thermal stability is relevant for the product to withstand harsh processing conditions.
- a standard ‘steam box’ assay was performed. Fusion proteins were dried with a SpeedVacTM vacuum concentrator and exposed to conditions mimicking formulations of feed products. Fusion proteins that were dried and treated with steam could then be compared to fusion proteins that were only dried.
- the melting temperature of the fusion proteins were measured on a Panta Prometheus system.
- Figure 7 shows the retained binding affinity of fusion proteins in percentage after treatment with ‘Steam box’.
- Table 2 shows the melting temperature and degree of refolding for 10 fusion proteins.
- This assay evaluates the effectiveness of single-domain antibodies of the invention on limiting the invasion of Eimeria spp. sporozoites in a cell culture model.
- the present assay involves incubating the antibodies at a concentration of 80 pg/mL with freshly prepared Eimeria tenella sporozoites for a period of 8 hours. While the assay described below was performed using these parameters, it can also be performed using different Eimeria species such as E. acervulina or E. maxima, and using other single-domain antibody concentrations or other incubation times.
- Each single-domain antibody is tested for efficacy against cell invasion separately by one or more Eimeria species.
- sporozoites of E. tenella were used, along with three different single-domain antibodies at concentration of 80 pg/mLm, and with a Monensin (a carboxylic polyether ionophore) as a positive control and a no-additive negative control.
- the antibodies can be diluted in cell culture media or any standard buffer such as PBS with a pH in the range of 5-9. Media or solutions containing proteases should be avoided.
- MDBK cells kidney epithelial cells
- the mixtures (0.5 mL) are transferred onto fresh cultures of MDBK cells (kidney epithelial cells) in 48-well plates and incubated for ⁇ 24 h (41 °C and 5% CO2).
- MDBK cells kidney epithelial cells
- An appropriate number of freshly excysted Eimeria sporozoites used in this step can vary depending e.g. on the species and may, for example, be in the range of about 1 ,000 to 10,000, such as about 2,500-10,000. In the case of E. tenella sporozoites in the present example, 2,500 sporozoites were used.
- An anti-E/meria monoclonal antibody (primary) is added to each well and allowed to react for 24 h, after which the plates are washed twice, and a secondary antibody (FITC- labeled) is added and incubated for 6 h followed by a final washing step.
- the number of infected cells is then differentially counted using a Keyence BZ- X810 system and the percent invasion is calculated for each well.
- UUC uninfected untreated control
- the UUC and the infected untreated control groups may e.g. include 6 replicates of 7 birds/replicate, and groups receiving single-domain antibodies or fusion proteins may e.g. include 8 replicates of 7 birds/replicate.
- Allocation of treatments to cages is random and birds are randomly allocated to each cage so the average body weight per cage is similar for all the cages.
- the chosen number of birds per replicate (e.g. 7) are randomly assigned to each cage per treatment group on the day of set-up and reduced to five birds per cage on the day of challenge to ensure homologous weights per group.
- Coccidiosis is induced by experimental infection of chickens, for example Ross 308 male broiler chickens, via oral gavage, with a field strain of pure Eimeria sp., for example E. tenella, E. maxima or E. acervulina, on day 7 of age, while UUC birds are sham inoculated.
- Treatment efficacy may be assessed by evaluation of different parameters, for example monitoring intestinal lesion scores and oocyst shedding as primary parameters, and body weight gain and feed intake as secondary parameters. Mortality is monitored throughout the trial to adjust the performance parameters.
- OPG oocysts per gram feces
- Trials may be performed for a total of e.g. 16 days, where the trial may be divided up into trial periods, for example day 0-7, day 7-14, day 14-16, day 7-16 and day 0-16 in the case of a 16-day trial.
- Average daily gain (ADG), average daily feed intake (ADFI) and the ADFI/ADG ratio may be calculated for all trial periods in the 16-day trial.
- OPG counts and lesion counts may be made on selected days, for example on one or more of days 12, 13, 14, 15 and 16 in the case of a 16-day trial.
- Comparison of measured parameters is conducted among i) an infected untreated control (IUC), and ii) one or more groups receiving the single-domain antibodies.
- IUC infected untreated control
- Single-domain antibodies may suitably be added to a European (wheat based) basal diet, formulated based on Ross 2022 recommendations, and provided to birds ad libitum throughout the trial (from 0-16 days of age).
- Feed mixing for the basal diet may be performed by spraying liquid containing the single-domain antibodies on feed pellets after pelleting and cooling.
- the antibodies may for example be diluted in plain water to obtain a spraying volume of approximately 2-2.5 ml/kg of pellets, with a batch volume of about 200 ml. This may be used for spraying on e.g. 75-100 kg of pellets, for example using a paddle mixer and a laboratory hose pump.
- a suitable dose of single-domain antibody or fusion protein may e.g. be determined on the basis of in vitro trials, by dose range finding studies, and/or based on the results of initial trials.
- Example 9 An in vivo proof-of-principle study was performed using the protocol described in Example 9.
- chickens were infected with E. acervulina, and the number of lesions in an infected control group and in a group treated with the single-domain antibody of SEQ ID NO: 25 was determined on day 14.
- the single-domain antibody was administered applied on feed pellets which were provided to the birds ad libitum as described in Example 9.
- the results, expressed as numbers of birds in the two groups with 0, 1 , 2 or 3 lesions per bird, are provided in Table 4.
- Table 4 Lesion scores in in vivo trial with chickens infected with E. acervulina
Abstract
The present invention relates to single-domain antibodies having a binding affinity for the antigen microneme protein 2 (MIC2) of Eimeria parasites; to fusion proteins comprising multimer components of the single-domain antibodies; and to the use of the fusion proteins for the prevention of coccidiosis in poultry.
Description
FUSION PROTEINS AND THEIR USE AGAINST EIMERIA
Reference to a Sequence Listing
This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
Background of the Invention
Coccidiosis in poultry production is a US$10 billion problem. Via malnutrition, decreased weight-gain, and chicken mortality, the poultry industry is on average globally losing substantial revenue.
The coccidiosis disease is caused by a genus of parasites known as Eimeria spp. The parasites infect new chicken hosts via the natural foraging habit of pecking the ground. During foraging, the animals pick up parasite oocysts, which then sporulate in the intestines of the chicken. In this sporulated stage, the parasite is described as a ‘sporozoite’. The sporozoite then move to the section of the gut where it may proliferate. The parasite invades a single chicken cell in the gut wall of the chicken intestine. Inside the chicken cell, the parasite multiplies before rupturing the cell. The increased number of parasites now invade other cells of the chicken gut, proliferating rapidly. In this way, the parasite multiplies until it can start creating new oocysts. These oocysts, or parasite eggs, are then shed via the chicken excrement which is then pecked at by another chicken and hereby also become infected. Cascading through the chicken population, a single parasite oocyst can infect an entire barn of birds rapidly.
Nine species of the intracellular protozoan parasite of the apicomplexan genus Eimeria causes avian coccidiosis. Among the nine Eimeria species infecting the gastrointestinal tracts of industrial chickens, three species are the primary focus of the invention: E. tenella, E. acervulina, and E. maxima. These are particularly prominent on farms and have different regions of the chicken intestines as invasion target, where they destroy intestinal epithelial cells causing severe damage to the intestinal wall of the chicken.
As a consequence of the widespread prophylactic use of anticoccidial agents, antimicrobial resistance is a growing and an inevitable problem in the poultry industry. New methods to control, prevent, or treat Eimeria parasitic infections in the intensively reared poultry industry are highly needed. An object of the invention is the development of new methods and agents which do not contribute to current antimicrobial resistance. The object of the present invention is to provide single-domain antibodies with high binding affinity for the antigen microneme protein 2 (MIC2).
MIC2 antigens are transmembrane proteins secreted by micronemes to the surface of the anterior region of the parasite in the sporozoite life stage where cell invasion occurs. MIC2 antigens are highly conserved proteins in the Eimeria parasite and are important in multiple
interactions, including motility and adherence, between the parasite and the host cell during intestinal epithelial host cell invasion. By blocking the MIC2 antigen, the sporozoite invasion of intestinal epithelial host cells can be inhibited.
A previous study has demonstrated the binding of polyclonal antibodies against EtMIC2 on the surface of live E. tenella sporozoites (M. Yan et al., “Molecular characterization and protective efficacy of the microneme 2 protein from Eimeria tenella," Parasite, vol. 25, 2018, doi: 10.1051/parasite/2018061).
WO2020/234642 discloses polypeptides comprising at least one variable region fragment of a heavy chain antibody (VHH), which specifically binds a disease-causing agent.
The object of the present invention is to provide single-domain antibodies with high binding affinity for the antigen microneme protein 2 (MIC2).
Field of the Invention
The present invention relates to single-domain antibodies having a binding affinity for the antigen microneme protein 2 (MIC2); to polynucleotides encoding the single-domain antibodies; to nucleic acid constructs; to host cells comprising the polynucleotides; to fusion proteins comprising multimer components of the single-domain antibodies; and to the use of the fusion proteins for binding to MIC2 for the prevention of coccidiosis in poultry.
Summary of the Invention
The present invention provides single-domain antibodies (sdAbs) having binding affinity for the antigen microneme protein 2 (MIC2); to fusion proteins comprising two or more sdAbs and to polynucleotides encoding the single-domain antibodies and/or fusion proteins.
An aspect of the invention is directed to a polypeptide selected from the group consisting of:
(a) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 1 ;
(b) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 5;
(c) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 9;
(d) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 13;
(e) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 17;
(f) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 21 ;
(g) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 25;
(h) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 29;
(i) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 33;
(j) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 37;
(k) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 41 ;
(l) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 45;
(m) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 49;
(n) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 53;
(o) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 82;
(p) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 86;
(q) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 90;
(r) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 94;
(s) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 98;
(t) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 102;
(u) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 106;
(v) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 110;
(w) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 114;
(x) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 118;
(y) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 122;
(z) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 126;
(aa) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 130;
(ab) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 134;
(ac) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 138;
(ad) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 142;
(ae) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 146;
(af) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 150;
(ag) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 154;
(ah) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 158;
(ai) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 162;
(aj) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 166;
(ak) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 170;
(al) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 174;
(am) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 178;
(an) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 182;
(ao) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 186;
(ap) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 190;
(aq) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 194;
(ar) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 198;
(as) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 202;
(at) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 206;
(au) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 210;
(av) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 214;
(aw) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 218;
(ax) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 222;
(ay) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 226;
(az) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 230;
(ba) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 234;
(bb) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 238;
(be) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 242;
(bd) a single-domain antibody encoded by a polynucleotide having at least 80% sequence identity to the coding sequence of any one of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; preferably SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, more preferably SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53;
(be) a single-domain antibody derived from any one of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; preferably SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, more preferably SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53 by having 1-20 alterations (e.g., substitutions, deletions and/or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 alterations, in particular substitutions; and
(bf) a single-domain antibody derived from the single-domain antibody of (a), (b), (c), (d), (e),
(f). (g). (h), (i), 0), (k), (I), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), (x), (y), (z), (aa), (ab), (ac),
(ad), (ae), (af), (ag), (ah), (ai), (aj), (ak), (al), (am), (an), (ao), (ap), (aq), (ar), (as), (at), (au), (av), (aw), (ax), (ay), (az), (ba), (bb) or (be), wherein the N- and/or C-terminal end has been extended by addition of one or more amino acids. In a preferred embodiment, this aspect of the invention is directed to a single-domain antibody having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53.
The single-domain antibody preferably has binding affinity for the antigen microneme protein 2 (MIC2).
A second aspect of the invention is directed to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ
ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:57, SEQ
ID NO:58, SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ
ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ
ID NQ:70 and SEQ ID NO:71.
A further aspect of the invention is directed to a single-domain antibody having a binding affinity for the antigen microneme protein 2 (MIC2), such as a binding affinity for one or more of MIC2 from Eimeria tenella (EtMIC2), MIC2 from Eimeria maxima (EmMIC2), and MIC2 from Eimeria acervulina (EaMIC2). A further aspect of the invention is directed to a single-domain antibody or a fusion protein comprising two or more sdAbs of the invention for preventing, treating or reducing the likelihood of an Eimeria infection in an animal such as poultry, comprising administering to the animal a single-domain antibody or fusion protein of the invention and further comprising a reduced dose or frequency of a coccidostat compared to a conventional dose or frequency.
A further aspect of the invention is directed to a fusion protein comprising two or more sdAbs of the invention.
A further aspect of the invention is directed to a composition comprising an sdAb of the invention. Similarly, an aspect of the invention is directed to a composition comprising the fusion protein of the invention. The invention further relates to a granule, which comprises: (a) a core comprising the single-domain antibody of the invention or the fusion protein of the invention, and optionally, (b) a coating consisting of one or more layer(s) surrounding the core. A related further aspect of the invention is directed to a granule, which comprises: (a) a core, and (b) a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises the singledomain antibody of the invention or the fusion protein of the invention. A further aspect is directed to a liquid composition comprising the single-domain antibody of the invention or the fusion protein of the invention, and a stabilizer.
A key aspect of the invention is directed to use of a fusion protein of the invention or of an sdAb of the invention for the preparation of an animal feed additive or animal feed. Related aspects of the invention are directed to a feed additive comprising an sdAb of the invention or the fusion protein of the invention. A further related aspect of the invention is directed to a feed comprising a feed additive of the invention.
A further key aspect of the invention is directed to a method of reducing the likelihood of the spread of Eimeria comprising the use of a granule, composition, feed additive, or feed of the invention. A further aspect of the invention is directed to a method of increasing the food conversion ratio (FCR) of an animal or the body weight gain (BWG) of an animal. A still further aspect of the invention is directed to a method of treating or preventing coccidiosis in poultry comprising the use of a granule, composition, feed additive, or feed of the invention.
Similarly, an aspect of the invention is directed to use of a fusion protein of the invention or an sdAb of the invention for the preparation of a medicament for the prevention or treatment of coccidiosis in poultry, and to a fusion protein of the invention or an sdAb of the invention for use as a medicament, e.g. for the prevention or treatment of coccidiosis in poultry. A further aspect of the invention is directed to a method of reducing antimicrobial resistance in animals, such as in poultry farming, said method comprising administering to said animal a feed or feed additive comprising a single-domain antibody or fusion protein of the invention. This method of reducing antimicrobial resistance suitably involves blocking binding sites of virulence factors, thereby disabling the virulence factors from binding to host cell receptors, thereby preventing pathogens from establishing an infection in the animals. A related aspect is directed to a method of preventing, treating or reducing the likelihood of an Eimeria infection which has a reduced proposentity to induce AMR.
The invention is furthermore directed to a method or preventing, treating or reducing the likelihood of an Eimeria infection in an animal such as poultry, comprising administering to the animal a single-domain antibody or fusion protein of the invention and further comprising a reduced dose or frequency of a coccidostat compared to a conventional dose or frequency.
The invention is furthermore directed to a method of reducing parasite invasion or reducing the risk of infections in a pet, such as but not limited to a dog, cat, bird, or rabbit.
The invention is furthermore directed to a polynucleotide encoding the single-domain antibody of the invention or fusion protein of the invention; to a nucleic acid construct or expression vector comprising the polynucleotide encoding the single-domain antibody of the invention or fusion protein of the invention, operably linked to one or more control sequences that direct the production of the single-domain antibody in an expression host; to a recombinant host cell comprising the nucleic acid construct or expression vector of the invention; and to a method of producing a single-domain antibody or fusion protein, comprising cultivating the recombinant host cell of the invention under conditions conducive for production of the single-domain antibody or fusion protein.
The Eimeria parasite is the causative agent behind coccidiosis in chickens and is a huge problem in the poultry industry. Low productivity and expenses for treatment and preventive matters are large burdens for farmers. Prevention and treatment with antimicrobial agents have led to increased resistance development against anticoccidial agents in Eimeria species. Fusion proteins comprising single-domain antibodies of the invention bind specifically to virulence factors of pathogenic microorganisms and thereby inactivate their virulence. Fusion proteins comprising single-domain antibodies impose a low selection pressure, do not favour resistance development in the microorganisms, or interact with the beneficial commensals in the gastrointestinal tract as antimicrobial agents do. For the three specific antigens of the Eimeria species, MIC2 is identified for blocking Eimeria adherence and cell invasion. Fusion proteins comprising single-domain antibodies of the invention have been constructed to bind specifically to MIC2 and to be mixed with or formulated within animal feed or an animal feed additive. It is alternatively formulated as a coccidiostat product.
The benefits of the fusion proteins comprising single-domain antibodies of the invention include:
- strengthening the gut microbiome and reducing the risk of gastrointestinal infections in hosts;
- high specificity for Eimeria antigens, avoiding interaction with the beneficial bacteria of the host;
- exerting their function extracellularly and thereby only imposing a small selection pressure on the Eimeria parasite, thereby also being effective against resistant variants;
- given that the functional site involved in the molecular virulence mechanism, where the fusion proteins comprising single-domain antibodies of the invention bind, is highly conserved, in the event of escape mutation, the Eimeria parasite will lose affinity for the natural receptor;
- they do not have a traditional bacteriocidal or bacteriostatic effect;
- the fusion proteins comprising single-domain antibodies of the invention are binding proteins suitable as food ingredients and feed additives.
Brief Description of the Figures
Figure 1 shows the alignment of MIC2 amino acid sequences using MUSCLE (version 3.8.1551) multiple sequence alignment algorithm. Grey bar over the consensus sequence indicates the level of conservation between sequences with dark grey = 100% consensus and white = 0% consensus. Conserved amino acids are marked in grey.
Figure 2 shows four different binding molecules related to the term ‘antibody’. Singledomain antibodies (sdAbs) are derived from the VHH-region of the camelid heavy-chain antibody. Fusion proteins are then derived from sdAbs by joining two sdAbs directly or through a linker.
Figure 3 illustrates the binding affinity of purified sdAbs against target MIC2 antigen (Table 1) evaluated via a standard DELFIA assay. TRF means time resolved fluorescence.
Figure 4 illustrates the alignment of sdAb amino acid sequences using MUSCLE multiple sequence alignment algorithm. Grey bar over the consensus sequence indicates level of conservation between sequences with dark grey = 100% consensus and white = 0% consensus. Conserved amino acids marked in grey.
Figure 5 illustrates the comparison in binding affinity between selected sdAbs and corresponding fusion proteins. A standard DELFIA assay was used (Figure 6). Note that the time resolved fluorescence (TRF) signal cannot be compared directly to Figure 3 due to a lower concentration of antibody (50 nM) in this assay.
Figure 6 illustrates a standard DELFIA assay to observe cross-reactivity of fusion proteins (a high concentration (1000 nM))
Figure 7 illustrates the retained binding affinity of fusion proteins in percentage after treatment with ‘Steam box’, a measurement of thermostability.
Figure 8 illustrates the binding of fusion proteins to homogenized Eimeria oocysts. Measured with ELISA. Background absorbance for each fusion protein without presence of oocyst was subtracted from each measurement.
Summary of the Sequences
SEQ ID NO:1 is the sequence of a single-domain antibody.
SEQ ID NO:2 is the sequence of a CDR1 region, found at least in SEQ ID NO:1.
SEQ ID NO:3 is the sequence of a CDR2 region, found at least in SEQ ID NO:1.
SEQ ID NO:4 is the sequence of a CDR3 region, found at least in SEQ ID NO:1.
SEQ ID NO:5 is the sequence of a single-domain antibody.
SEQ ID NO:6 is the sequence of a CDR1 region, found at least in SEQ ID NO:5.
SEQ ID NO:7 is the sequence of a CDR2 region, found at least in SEQ ID NO:5.
SEQ ID NO:8 is the sequence of a CDR3 region, found at least in SEQ ID NO:5.
SEQ ID NO:9 is the sequence of a single-domain antibody.
SEQ ID NQ:10 is the sequence of a CDR1 region, found at least in SEQ ID NO:9.
SEQ ID NO:11 is the sequence of a CDR2 region, found at least in SEQ ID NO:9.
SEQ ID NO:12 is the sequence of a CDR3 region, found at least in SEQ ID NO:9.
SEQ ID NO: 13 is the sequence of a single-domain antibody.
SEQ ID NO:14 is the sequence of a CDR1 region, found at least in SEQ ID NO:13.
SEQ ID NO:15 is the sequence of a CDR2 region, found at least in SEQ ID NO:13.
SEQ ID NO: 16 is the sequence of a CDR3 region, found at least in SEQ ID NO: 13.
SEQ ID NO: 17 is the sequence of a single-domain antibody.
SEQ ID NO:18 is the sequence of a CDR1 region, found at least in SEQ ID NO:17.
SEQ ID NO:19 is the sequence of a CDR2 region, found at least in SEQ ID NO:17.
SEQ ID NQ:20 is the sequence of a CDR3 region, found at least in SEQ ID NO:17.
SEQ ID NO:21 is the sequence of a single-domain antibody.
SEQ ID NO:22 is the sequence of a CDR1 region, found at least in SEQ ID NO:21.
SEQ ID NO:23 is the sequence of a CDR2 region, found at least in SEQ ID NO:21.
SEQ ID NO:24 is the sequence of a CDR3 region, found at least in SEQ ID NO:21.
SEQ ID NO:25 is the sequence of a single-domain antibody.
SEQ ID NO:26 is the sequence of a CDR1 region, found at least in SEQ ID NO:25.
SEQ ID NO:27 is the sequence of a CDR2 region, found at least in SEQ ID NO:25.
SEQ ID NO:28 is the sequence of a CDR3 region, found at least in SEQ ID NO:25.
SEQ ID NO:29 is the sequence of a single-domain antibody.
SEQ ID NQ:30 is the sequence of a CDR1 region, found at least in SEQ ID NO:29.
SEQ ID NO:31 is the sequence of a CDR2 region, found at least in SEQ ID NO:29.
SEQ ID NO:32 is the sequence of a CDR3 region, found at least in SEQ ID NO:29.
SEQ ID NO:33 is the sequence of a single-domain antibody.
SEQ ID NO:34 is the sequence of a CDR1 region, found at least in SEQ ID NO:33.
SEQ ID NO:35 is the sequence of a CDR2 region, found at least in SEQ ID NO:33.
SEQ ID NO:36 is the sequence of a CDR3 region, found at least in SEQ ID NO:33.
SEQ ID NO:37 is the sequence of a single-domain antibody.
SEQ ID NO:38 is the sequence of a CDR1 region, found at least in SEQ ID NO:37.
SEQ ID NO:39 is the sequence of a CDR2 region, found at least in SEQ ID NO:37.
SEQ ID NQ:40 is the sequence of a CDR3 region, found at least in SEQ ID NO:37.
SEQ ID NO:41 is the sequence of a single-domain antibody.
SEQ ID NO:42 is the sequence of a CDR1 region, found at least in SEQ ID NO:41.
SEQ ID NO:43 is the sequence of a CDR2 region, found at least in SEQ ID NO:41.
SEQ ID NO:44 is the sequence of a CDR3 region, found at least in SEQ ID NO:41.
SEQ ID NO:45 is the sequence of a single-domain antibody.
SEQ ID NO:46 is the sequence of a CDR1 region, found at least in SEQ ID NO:45.
SEQ ID NO:47 is the sequence of a CDR2 region, found at least in SEQ ID NO:45.
SEQ ID NO:48 is the sequence of a CDR3 region, found at least in SEQ ID NO:45.
SEQ ID NO:49 is the sequence of a single-domain antibody.
SEQ ID NQ:50 is the sequence of a CDR1 region, found at least in SEQ ID NO:49.
SEQ ID NO:51 is the sequence of a CDR2 region, found at least in SEQ ID NO:49.
SEQ ID NO:52 is the sequence of a CDR3 region, found at least in SEQ ID NO:49.
SEQ ID NO:53 is the sequence of a single-domain antibody.
SEQ ID NO:54 is the sequence of a CDR1 region, found at least in SEQ ID NO:53.
SEQ ID NO:55 is the sequence of a CDR2 region, found at least in SEQ ID NO:53.
SEQ ID NO:56 is the sequence of a CDR3 region, found at least in SEQ ID NO:53.
SEQ ID NO:57 is the sequence of a CDR3 region of Family 8.
SEQ ID NO:58 is the sequence of a CDR3 region of Family 8.
SEQ ID NO:59 is the sequence of a CDR3 region of Family 10.
SEQ ID NO:60 is the sequence of a CDR3 region of Family 13.
SEQ ID NO:61 is the sequence of a CDR3 region of Family 13.
SEQ ID NO:62 is the sequence of a CDR3 region of Family 21.
SEQ ID NO:63 is the sequence of a CDR3 region of Family 21.
SEQ ID NO:64 is the sequence of a CDR3 region of Family 21.
SEQ ID NO:65 is the sequence of a CDR3 region of Family 23.
SEQ ID NO:66 is the sequence of a CDR3 region of Family 23. SEQ ID NO:67 is the sequence of a CDR3 region of Family 23. SEQ ID NO:68 is the sequence of a CDR3 region of Family 23. SEQ ID NO:69 is the sequence of a CDR3 region of Family 23. SEQ ID NQ:70 is the sequence of a CDR3 region of Family 23. SEQ ID NO:71 is the sequence of a CDR3 region of Family 23. SEQ ID NO:72 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:1 . SEQ ID NO:73 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:33. SEQ ID NO:74 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:37.
SEQ ID NO:75 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:45. SEQ ID NO:76 is the sequence of a fusion protein compring a homodimer of SEQ ID NO: 13. SEQ ID NO:77 is the sequence of a fusion protein compring a homodimer of SEQ ID NO: 17. SEQ ID NO:78 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:53. SEQ ID NO:79 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:21. SEQ ID NQ:80 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:25. SEQ ID NO:81 is the sequence of a fusion protein compring a homodimer of SEQ ID NO:9. SEQ ID NO:82 is the sequence of a single-domain antibody.
SEQ ID NO:83 is the sequence of a CDR1 region, found at least in SEQ ID NO:82.
SEQ ID NO:84 is the sequence of a CDR2 region, found at least in SEQ ID NO:82.
SEQ ID NO:85 is the sequence of a CDR3 region, found at least in SEQ ID NO:82.
SEQ ID NO:86 is the sequence of a single-domain antibody.
SEQ ID NO:87 is the sequence of a CDR1 region, found at least in SEQ ID NO:86.
SEQ ID NO:88 is the sequence of a CDR2 region, found at least in SEQ ID NO:86.
SEQ ID NO:89 is the sequence of a CDR3 region, found at least in SEQ ID NO:86.
SEQ ID NQ:90 is the sequence of a single-domain antibody.
SEQ ID NO:91 is the sequence of a CDR1 region, found at least in SEQ ID NQ:90.
SEQ ID NO:92 is the sequence of a CDR2 region, found at least in SEQ ID NQ:90.
SEQ ID NO:93 is the sequence of a CDR3 region, found at least in SEQ ID NQ:90.
SEQ ID NO:94 is the sequence of a single-domain antibody.
SEQ ID NO:95 is the sequence of a CDR1 region, found at least in SEQ ID NO:94.
SEQ ID NO:96 is the sequence of a CDR2 region, found at least in SEQ ID NO:94.
SEQ ID NO:97 is the sequence of a CDR3 region, found at least in SEQ ID NO:94.
SEQ ID NO:98 is the sequence of a single-domain antibody.
SEQ ID NO:99 is the sequence of a CDR1 region, found at least in SEQ ID NO:98.
SEQ ID NO: 100 is the sequence of a CDR2 region, found at least in SEQ ID NO:98.
SEQ ID NQ:101 is the sequence of a CDR3 region, found at least in SEQ ID NO:98.
SEQ ID NQ:102 is the sequence of a single-domain antibody.
SEQ ID NQ:103 is the sequence of a CDR1 region, found at least in SEQ ID NQ:102.
SEQ ID NQ:104 is the sequence of a CDR2 region, found at least in SEQ ID NQ:102.
SEQ ID NQ:105 is the sequence of a CDR3 region, found at least in SEQ ID NQ:102.
SEQ ID NQ:106 is the sequence of a single-domain antibody.
SEQ ID NQ:107 is the sequence of a CDR1 region, found at least in SEQ ID NQ:106.
SEQ ID NQ:108 is the sequence of a CDR2 region, found at least in SEQ ID NQ:106.
SEQ ID NQ:109 is the sequence of a CDR3 region, found at least in SEQ ID NQ:106.
SEQ ID NQ:110 is the sequence of a single-domain antibody.
SEQ ID NO:111 is the sequence of a CDR1 region, found at least in SEQ ID NQ:110.
SEQ ID NO:112 is the sequence of a CDR2 region, found at least in SEQ ID NQ:110.
SEQ ID NO:113 is the sequence of a CDR3 region, found at least in SEQ ID NQ:110.
SEQ ID NO:114 is the sequence of a single-domain antibody.
SEQ ID NO:115 is the sequence of a CDR1 region, found at least in SEQ ID NO:114.
SEQ ID NO:116 is the sequence of a CDR2 region, found at least in SEQ ID NO:114.
SEQ ID NO:117 is the sequence of a CDR3 region, found at least in SEQ ID NO:114.
SEQ ID NO:118 is the sequence of a single-domain antibody.
SEQ ID NO:119 is the sequence of a CDR1 region, found at least in SEQ ID NO:118.
SEQ ID NQ:120 is the sequence of a CDR2 region, found at least in SEQ ID NO:118.
SEQ ID NO:121 is the sequence of a CDR3 region, found at least in SEQ ID NO:118.
SEQ ID NO:122 is the sequence of a single-domain antibody.
SEQ ID NO:123 is the sequence of a CDR1 region, found at least in SEQ ID NO:122.
SEQ ID NO:124 is the sequence of a CDR2 region, found at least in SEQ ID NO:122.
SEQ ID NO:125 is the sequence of a CDR3 region, found at least in SEQ ID NO:122.
SEQ ID NO: 126 is the sequence of a single-domain antibody.
SEQ ID NO:127 is the sequence of a CDR1 region, found at least in SEQ ID NO:126.
SEQ ID NO:128 is the sequence of a CDR2 region, found at least in SEQ ID NO:126.
SEQ ID NO:129 is the sequence of a CDR3 region, found at least in SEQ ID NO:126.
SEQ ID NQ:130 is the sequence of a single-domain antibody.
SEQ ID NO:131 is the sequence of a CDR1 region, found at least in SEQ ID NQ:130.
SEQ ID NO:132 is the sequence of a CDR2 region, found at least in SEQ ID NQ:130.
SEQ ID NO:133 is the sequence of a CDR3 region, found at least in SEQ ID NQ:130.
SEQ ID NO: 134 is the sequence of a single-domain antibody.
SEQ ID NO:135 is the sequence of a CDR1 region, found at least in SEQ ID NO:134.
SEQ ID NO:136 is the sequence of a CDR2 region, found at least in SEQ ID NO:134.
SEQ ID NO:137 is the sequence of a CDR3 region, found at least in SEQ ID NO:134.
SEQ ID NO: 138 is the sequence of a single-domain antibody.
SEQ ID NO:139 is the sequence of a CDR1 region, found at least in SEQ ID NO:138.
SEQ ID NQ:140 is the sequence of a CDR2 region, found at least in SEQ ID NO:138.
SEQ ID NO:141 is the sequence of a CDR3 region, found at least in SEQ ID NO:138.
SEQ ID NO:142 is the sequence of a single-domain antibody.
SEQ ID NO:143 is the sequence of a CDR1 region, found at least in SEQ ID NO:142.
SEQ ID NO:144 is the sequence of a CDR2 region, found at least in SEQ ID NO:142.
SEQ ID NO:145 is the sequence of a CDR3 region, found at least in SEQ ID NO:142.
SEQ ID NO:146 is the sequence of a single-domain antibody.
SEQ ID NO:147 is the sequence of a CDR1 region, found at least in SEQ ID NO:146.
SEQ ID NO:148 is the sequence of a CDR2 region, found at least in SEQ ID NO:146.
SEQ ID NO:149 is the sequence of a CDR3 region, found at least in SEQ ID NO:146.
SEQ ID NO: 150 is the sequence of a single-domain antibody.
SEQ ID NO:151 is the sequence of a CDR1 region, found at least in SEQ ID NQ:150.
SEQ ID NO:152 is the sequence of a CDR2 region, found at least in SEQ ID NQ:150.
SEQ ID NO:153 is the sequence of a CDR3 region, found at least in SEQ ID NQ:150.
SEQ ID NO: 154 is the sequence of a single-domain antibody.
SEQ ID NO:155 is the sequence of a CDR1 region, found at least in SEQ ID NO:154.
SEQ ID NO:156 is the sequence of a CDR2 region, found at least in SEQ ID NO:154.
SEQ ID NO:157 is the sequence of a CDR3 region, found at least in SEQ ID NO:154.
SEQ ID NO: 158 is the sequence of a single-domain antibody.
SEQ ID NO:159 is the sequence of a CDR1 region, found at least in SEQ ID NO:158.
SEQ ID NQ:160 is the sequence of a CDR2 region, found at least in SEQ ID NO:158.
SEQ ID NO:161 is the sequence of a CDR3 region, found at least in SEQ ID NO:158.
SEQ ID NO:162 is the sequence of a single-domain antibody.
SEQ ID NO:163 is the sequence of a CDR1 region, found at least in SEQ ID NO:162.
SEQ ID NO:164 is the sequence of a CDR2 region, found at least in SEQ ID NO:162.
SEQ ID NO:165 is the sequence of a CDR3 region, found at least in SEQ ID NO:162.
SEQ ID NO:166 is the sequence of a single-domain antibody.
SEQ ID NO:167 is the sequence of a CDR1 region, found at least in SEQ ID NO:166.
SEQ ID NO:168 is the sequence of a CDR2 region, found at least in SEQ ID NO:166.
SEQ ID NO:169 is the sequence of a CDR3 region, found at least in SEQ ID NO:166.
SEQ ID NO: 170 is the sequence of a single-domain antibody.
SEQ ID NO:171 is the sequence of a CDR1 region, found at least in SEQ ID NQ:170.
SEQ ID NO:172 is the sequence of a CDR2 region, found at least in SEQ ID NQ:170.
SEQ ID NO:173 is the sequence of a CDR3 region, found at least in SEQ ID NQ:170.
SEQ ID NO: 174 is the sequence of a single-domain antibody.
SEQ ID NO:175 is the sequence of a CDR1 region, found at least in SEQ ID NO:174.
SEQ ID NO:176 is the sequence of a CDR2 region, found at least in SEQ ID NO:174.
SEQ ID NO:177 is the sequence of a CDR3 region, found at least in SEQ ID NO:174.
SEQ ID NO:178 is the sequence of a single-domain antibody.
SEQ ID NO:179 is the sequence of a CDR1 region, found at least in SEQ ID NO:178.
SEQ ID NQ:180 is the sequence of a CDR2 region, found at least in SEQ ID NO:178.
SEQ ID NO:181 is the sequence of a CDR3 region, found at least in SEQ ID NO:178.
SEQ ID NO:182 is the sequence of a single-domain antibody.
SEQ ID NO:183 is the sequence of a CDR1 region, found at least in SEQ ID NO:182.
SEQ ID NO:184 is the sequence of a CDR2 region, found at least in SEQ ID NO:182.
SEQ ID NO:185 is the sequence of a CDR3 region, found at least in SEQ ID NO:182.
SEQ ID NO:186 is the sequence of a single-domain antibody.
SEQ ID NO:187 is the sequence of a CDR1 region, found at least in SEQ ID NO:186.
SEQ ID NO:188 is the sequence of a CDR2 region, found at least in SEQ ID NO:186.
SEQ ID NO:189 is the sequence of a CDR3 region, found at least in SEQ ID NO:186.
SEQ ID NQ:190 is the sequence of a single-domain antibody.
SEQ ID NO:191 is the sequence of a CDR1 region, found at least in SEQ ID NQ:190.
SEQ ID NO:192 is the sequence of a CDR2 region, found at least in SEQ ID NQ:190.
SEQ ID NO:193 is the sequence of a CDR3 region, found at least in SEQ ID NQ:190.
SEQ ID NO:194 is the sequence of a single-domain antibody.
SEQ ID NO:195 is the sequence of a CDR1 region, found at least in SEQ ID NO:194.
SEQ ID NO:196 is the sequence of a CDR2 region, found at least in SEQ ID NO:194.
SEQ ID NO:197 is the sequence of a CDR3 region, found at least in SEQ ID NO:194.
SEQ ID NO:198 is the sequence of a single-domain antibody.
SEQ ID NO:199 is the sequence of a CDR1 region, found at least in SEQ ID NO:198.
SEQ ID NQ:200 is the sequence of a CDR2 region, found at least in SEQ ID NO:198.
SEQ ID NQ:201 is the sequence of a CDR3 region, found at least in SEQ ID NO:198.
SEQ ID NQ:202 is the sequence of a single-domain antibody.
SEQ ID NQ:203 is the sequence of a CDR1 region, found at least in SEQ ID NQ:202.
SEQ ID NQ:204 is the sequence of a CDR2 region, found at least in SEQ ID NQ:202.
SEQ ID NQ:205 is the sequence of a CDR3 region, found at least in SEQ ID NQ:202.
SEQ ID NQ:206 is the sequence of a single-domain antibody.
SEQ ID NQ:207 is the sequence of a CDR1 region, found at least in SEQ ID NQ:206.
SEQ ID NQ:208 is the sequence of a CDR2 region, found at least in SEQ ID NQ:206.
SEQ ID NQ:209 is the sequence of a CDR3 region, found at least in SEQ ID NQ:206.
SEQ ID NQ:210 is the sequence of a single-domain antibody.
SEQ ID NO:211 is the sequence of a CDR1 region, found at least in SEQ ID NQ:210.
SEQ ID NO:212 is the sequence of a CDR2 region, found at least in SEQ ID NO:210.
SEQ ID NO:213 is the sequence of a CDR3 region, found at least in SEQ ID NO:210.
SEQ ID NO:214 is the sequence of a single-domain antibody.
SEQ ID NO:215 is the sequence of a CDR1 region, found at least in SEQ ID NO:214.
SEQ ID NO:216 is the sequence of a CDR2 region, found at least in SEQ ID NO:214.
SEQ ID NO:217 is the sequence of a CDR3 region, found at least in SEQ ID NO:214.
SEQ ID NO:218 is the sequence of a single-domain antibody.
SEQ ID NO:219 is the sequence of a CDR1 region, found at least in SEQ ID NO:218.
SEQ ID NQ:220 is the sequence of a CDR2 region, found at least in SEQ ID NO:218.
SEQ ID NO:221 is the sequence of a CDR3 region, found at least in SEQ ID NO:218.
SEQ ID NO:222 is the sequence of a single-domain antibody.
SEQ ID NO:223 is the sequence of a CDR1 region, found at least in SEQ ID NO:222.
SEQ ID NO:224 is the sequence of a CDR2 region, found at least in SEQ ID NO:222.
SEQ ID NO:225 is the sequence of a CDR3 region, found at least in SEQ ID NO:222.
SEQ ID NO:226 is the sequence of a single-domain antibody.
SEQ ID NO:227 is the sequence of a CDR1 region, found at least in SEQ ID NO:226.
SEQ ID NO:228 is the sequence of a CDR2 region, found at least in SEQ ID NO:226.
SEQ ID NO:229 is the sequence of a CDR3 region, found at least in SEQ ID NO:226.
SEQ ID NQ:230 is the sequence of a single-domain antibody.
SEQ ID NO:231 is the sequence of a CDR1 region, found at least in SEQ ID NQ:230.
SEQ ID NO:232 is the sequence of a CDR2 region, found at least in SEQ ID NQ:230.
SEQ ID NO:233 is the sequence of a CDR3 region, found at least in SEQ ID NQ:230.
SEQ ID NO:234 is the sequence of a single-domain antibody.
SEQ ID NO:235 is the sequence of a CDR1 region, found at least in SEQ ID NO:234.
SEQ ID NO:236 is the sequence of a CDR2 region, found at least in SEQ ID NO:234.
SEQ ID NO:237 is the sequence of a CDR3 region, found at least in SEQ ID NO:234.
SEQ ID NO:238 is the sequence of a single-domain antibody.
SEQ ID NO:239 is the sequence of a CDR1 region, found at least in SEQ ID NO:238.
SEQ ID NQ:240 is the sequence of a CDR2 region, found at least in SEQ ID NO:238.
SEQ ID NO:241 is the sequence of a CDR3 region, found at least in SEQ ID NO:238.
SEQ ID NO:242 is the sequence of a single-domain antibody.
SEQ ID NO:243 is the sequence of a CDR1 region, found at least in SEQ ID NO:242.
SEQ ID NO:244 is the sequence of a CDR2 region, found at least in SEQ ID NO:242.
SEQ ID NO:245 is the sequence of a CDR3 region, found at least in SEQ ID NO:242.
Definitions
In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.
Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Antibody: The term ‘antibody’ commonly refers to large human antibodies. According to the invention, the smaller single-domain antibodies (sdAbs) have beneficial biochemical and cost of manufacturing properties. Single-domain antibodies, also known as ‘nanobodies’ or ‘VHHs’, are small binding molecules derived from camelid antibodies.
Animal: The term “animal” refers to all animals except humans. Examples of animals are non-ruminants, and ruminants. Ruminant animals include, for example, animals such as sheep, goats, cattle, e.g. beef cattle, cows, and young calves, deer, yank, camel, llama and kangaroo. Non-ruminant animals include mono-gastric animals, e.g. pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); cats; dogs; horses (including but not limited to hotbloods, coldbloods and warm bloods), young calves; fish (including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish); and crustaceans (including but not limited to shrimps and prawns).
Animal feed: The term "animal feed" refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal. Animal feed for a monogastric animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbial, amino acids, and/or other feed ingredients (such as in a premix) whereas animal feed for ruminants generally comprises forage (including roughage and silage) and may further comprise concentrates as well as vitamins, minerals, enzymes direct fed microbial, amino acid, and/or other feed ingredients (such as in a premix).
Concentrates: The term "concentrates" means feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from e.g. corn, oats, rye, barley, wheat), oilseed press cake (e.g. from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).
cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
CDR region: The term ”CDR” means complementarity-determining region (in sdAbs there are three: CDR1 , CDR2 and CDR3). The CDR regions are part of the variable domain of an antibody, including the sdAbs of the present invention, and determine the antigen-binding specificity of the antibody. The CDR3 is the most variable of the CDRs and is a key determinant of antibody binding specificity.
Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a single-domain antibody. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
Control sequences: The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native (/.e., from the same gene) or heterologous (/.e., from a different gene) to the polynucleotide encoding the single-domain antibody, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a single-domain antibody.
Expression: The term “expression” means any step involved in the production of a singledomain antibody including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a single-domain antibody, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.
Family: The term ’’family” refers to sdAbs as classified by their CDR3 homologies.
Forage: The term "forage" as defined herein also includes roughage. Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof. Examples of forage plants are Alfalfa (lucerne), birdsfoot trefoil, brassica (e.g. kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g. alsike clover, red clover, subterranean clover, white clover), grass (e.g. Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, orchard grass, ryegrass, Timothy-grass), corn (maize), millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets. Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains), residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.
Fusion peptide: The term ’’fusion peptide” or “fusion single-domain antibody” is a singledomain antibody in which a first single-domain antibody is fused at the N-terminus and/or the C-terminus with a second single-domain antibody of the present invention. A fusion single-domain antibody may be produced by fusing a polynucleotide encoding a singledomain antibody of the present invention to a polynucleotide encoding another polypeptide, or by fusing two or more polynucleotides of the present invention together. Otherwise stated, the process involves extending one sdAbs coding sequences with the coding sequences of one or more polypeptides. Techniques for producing fusion single-domain antibodies are known in the art, and include ligating the coding sequences encoding the single-domain antibodies so that they are in frame and that expression of the fusion single-domain antibody is under control of the same promoter(s) and terminator.
Fusion single-domain antibodies may also be constructed using intein technology in which fusion single-domain antibodies are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusion single-domain antibody can further comprise a cleavage site between the two single-domain antibodies. Upon secretion of the fusion protein, the site is cleaved releasing the two single-domain antibodies. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen- Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward etal., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35- 48.
Homodimer : The term "homodimer" means a homo divalent protein construct and refers to a protein comprising a first peptide having a first binding specificity and a second peptide
having a second binding specificity connected by a linker, wherein the peptides are the same or substantially the same such as having at least 70% sequence identity.
Heterodimer : The term "heterodimer" means a hetero divalent protein construct and refers to a protein comprising a first peptide having a first binding specificity and a second peptide having a second binding specificity connected by a linker, wherein the peptides are different or substantially different such as having less than 70% sequence identity.
Heterologous: The term "heterologous" means, with respect to a host cell, that a singledomain antibody or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a single-domain antibody or nucleic acid, that a control sequence, e.g., promoter, of a single-domain antibody or nucleic acid is not naturally associated with the singledomain antibody or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature single-domain antibody.
Host Strain or Host Cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a single-domain antibody of the present invention has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the single-domain antibody of interest and/or fermenting saccharides. The term "host cell" includes protoplasts created from cells.
Improving: the term "Improving" means that one or more parameters influenced by an infection, such as Eimeria and Clostridium perfringens infections, are changing in a desireable direction. For example, in the broiler chickens with Eimeria and Clostridium perfringens infections, the body weight gain is increased, the body lesion is reduced and/or the expression of the antiinflammatory cytokines such as IL10 and IL8 is increased, etc.
Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation", or "transduction," as known in the art.
Isolated: The term “isolated” means a single-domain antibody, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated singledomain antibody, nucleic acid, cell, or other material is thus in a form that does not occur in nature. An isolated single-domain antibody includes, but is not limited to, a culture broth containing the secreted single-domain antibody expressed in a host cell.
Mature single-domain antibody: The term “mature single-domain antibody” means a single-domain antibody in its mature form following N-terminal and/or C-terminal processing (e.g., removal of signal peptide).
Mature single-domain antibody coding sequence: The term “mature single-domain antibody coding sequence” means a polynucleotide that encodes a mature single-domain antibody having MIC2 binding affinity.
MIC2: The term “MIC2” means the antigen microneme protein 2 from Eimeria, more precisely the Microneme protein 2 of the Eimeria spp. genus or equivalent homologs from other apicomplexan parasites, such as the MIC2-associated protein (M2AP) of the Toxoplasma spp. genus. Figure 1 shows the alignment of MIC2 amino acid sequences.
Native: The term "native" means a nucleic acid or single-domain antibody naturally occurring in a host cell.
Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a single-domain antibody. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerated, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.
Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.
Operably linked: The term "operably linked" means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.
Preventing: the term "preventing" means the prevention of the onset, recurrence, or spread, in whole or in part, of an infection such as Eimeria and Clostridium perfringens infections, or a symptom thereof.
Purified: The term “purified” means a nucleic acid, single-domain antibody, or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified single-domain antibody or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation). A purified nucleic acid or single-domain antibody is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, single-domain antibody, cell, nucleic acid, amino acid, or other specified material or component
that is present in a composition at a relative or absolute concentration that is higher than a starting composition.
In one aspect, the term "purified" as used herein refers to the single-domain antibody or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects the term "purified" refers to the single-domain antibody being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the single-domain antibody is separated from some of the soluble components of the organism and culture medium from which it is recovered. The single-domain antibody may be purified (/.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.
Accordingly, the single-domain antibody may be purified such that only minor amounts of other proteins, in particular other single-domain antibodies, are present. The term "purified" as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the single-domain antibody. The singledomain antibody may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced single-domain antibody. In one aspect, the single-domain antibody is at least 40% pure by weight of the total singledomain antibody material present in the preparation. In one aspect, the single-domain antibody is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total single-domain antibody material present in the preparation. As used herein a "substantially pure single-domain antibody" may denote a single-domain antibody preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1 %, and even most preferably at most 0.5% by weight of other single-domain antibody material with which the single-domain antibody is natively or recombinantly associated.
It is, therefore, preferred that the substantially pure single-domain antibody is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total singledomain antibody material present in the preparation. The single-domain antibody of the present invention is preferably in a substantially pure form {i.e., the preparation is essentially free of other single-domain antibody material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the single-domain antibody by well-known recombinant methods or by classical purification methods.
Recombinant: The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, single-domain antibody or vector that has been modified from its native state. Thus, for example, recombinant
cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.
Recover: The terms "recover" or “recovery” means the removal of a single-domain antibody from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the single-domain antibody from the whole fermentation broth, or from the cell-free fermentation broth, by single-domain antibody crystal harvest, by filtration, e.g., depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheed or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hyrdo cyclones or similar), or by precipitating the single-domain antibody and using relevant solid-liquid separation methods to harvest the single-domain antibody from the broth media by use of classification separation by particle sizes. Recovery encompasses isolation and/or purification of the single-domain antibody.
Roughage: The term "roughage" means dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste).
Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.
For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:
(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief
option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:
(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.
Treating: the term "treating" means alleviation, in whole or in part, of an infection, such as Eimeria and Clostridium perfringens infections, or a symptom thereof, or slowing, or halting of further progression or worsening of an infection.
Variant: The term “variant” means a single-domain antibody having MIC2 binding activity comprising a man-made mutation, i.e., a substitution, insertion (including extension), and/or deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.
Detailed Description of the Invention
Antibody engineering is a powerful technique to improve the effector function, stability, or utilize the binding abilities of antibodies in different contexts. The discovery that camelids (camels, llamas, alpacas etc.) naturally possess functional antibodies only consisting of heavy chains, initiated the development of the single-domain antibody technology. The camelid antibodies consist of two identical polypeptide chains each containing two constant domains (CH2 and CH3) and a single variable domain ( HH). The molecule is 2/3 the size of a human monoclonal antibody (mAb). In the context of fusion protein, the single-domain antibody is the recombinant variable domain ( HH) of camelid heavy-chain antibodies (hcAb), and, said more generally, a small stable binding protein. sdAbs have a unique structure and several advantageous functions compared to many other binding proteins and antibody formats, including high stability, high specificity, and affinity, good solubility, and low immunogenicity. Additionally, the relatively small size of 12-17 kD allows sdAbs to act in narrow or hidden regions where a human mAb would not fit. sdAbs have high stability and are very robust compared to many other proteins. They can resist chemical and thermal denaturation. sdAbs have the capacity of reversible refolding, which make them able to return to physiological conditions. They can be stored for months at 4 °C and even longer at -20°C and still maintain the antigen-binding capacity. The extended CDR3 loop on sdAbs enlarges flexibility and ability to bind to hidden antigens. sdAbs have good solubility since they
do not have a hydrophobic interaction, as human mAbs have, instead they have a hydrophilic side.
The low production costs and beneficial biochemical properties of sdAbs make them ideal candidates as sustainable functional food ingredients and feed additives. The fusion protein products are constructed of two sdAbs, such as two identical sdAbs, linked together by a protein linker, increasing their functional binding affinity due to increased avidity. The fusion proteins of the invention can be ingested orally as a feed additive because of the high stability of the product, combined with being active and binding extracellularly to virulence factors throughout the gastrointestinal tract. Ingestion of the fusion proteins of the invention reduces the risk of gastrointestinal infections and supports a healthy gut microbiome.
Single-domain antibodies having a binding affinity for the antigen microneme protein 2
The invention is directed to single-domain antibodies having a binding affinity for the antigen microneme protein 2 (MIC2A).
In a first aspect, the invention is directed to a single-domain antibody comprising a CDR3 region having at least 75% seguence identity to a seguence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28,
SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:58,
SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,
SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70 and SEQ ID NO:71. In one embodiment, the single-domain antibody comprises a CDR3 region having at least 80% seguence identity, such as at least 85% seguence identity, or at least 90% seguence identity, such as at least 95% seguence identity, such as 100% seguence identity, to a seguence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44,
SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 ,
SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,
SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70 and SEQ ID NO:71. In a preferred embodiment, the CDR3 region has at least 75% seguence identity, such as at least 80% seguence identity, such as at least 85% seguence identity, or at least 90% seguence identity, such as at least 95% seguence identity, such as 100% seguence identity, to a seguence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, and SEQ ID NO:56. In a more preferred embodiment, the CDR3 region has at least 75% seguence identity, such as at least 80% seguence identity, such as at least 85% seguence identity, or at least 90% seguence identity, such as at least 95% seguence identity, such as 100% seguence identity, to a seguence selected from the group consisting of SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:48 and SEQ ID NO:56.
In a suitable embodiment, the single-domain antibody comprises a CDR3 region having a sequence selected from the group consisting ofSEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44,
SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 ,
SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,
SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70 and SEQ ID NO:71 , such as a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, and SEQ ID NO:56, typically a sequence selected from the group consisting of SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:48 and SEQ ID NO:56.
The inventors have identified a number of unique sdAbs, which can be categorized according to their CDR3 regions. For instance, the sdAb comprising SEQ ID NO: 16 is categorized in Family 8 where there is high homology within the CDR3 region of the sdAb. These include SEQ ID NO: 57 and SEQ ID NO: 58. Accordingly, in one embodiment of the invention, the sdAb comprises a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 57 and SEQ ID NO: 58. In a more preferred embodiment, the CDR3 region has as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 57 and SEQ ID NO: 58.
Another family of interest are sdAbs of Family 10, which comprises SEQ ID NO:24 and SEQ ID NO: 59. Accordingly, in one embodiment of the invention, the sdAb comprises a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO: 59. In a more preferred embodiment, the CDR3 region has as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO: 59.
A further family of interest are sdAbs of Family 13, which comprises SEQ ID NO:28, SEQ ID NQ:60 and SEQ ID NO: 61. Accordingly, in one embodiment of the invention, the sdAb comprises a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NQ:60 and SEQ ID NO: 61 . In a more preferred embodiment, the CDR3 region has as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NQ:60 and SEQ ID NO: 61.
A further family of interest are sdAbs of Family 21 , which comprises SEQ ID NQ:20, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO: 64. Accordingly, in one embodiment of the invention, the sdAb comprises a CDR3 region having at least 75% sequence identity to a sequence selected
from the group consisting of SEQ ID NO:20, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO: 64. In a more preferred embodiment, the CDR3 region has as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO: 64.
A further family of interest are sdAbs of Family 23, which comprises SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70 and SEQ ID NO:71 . Accordingly, in one embodiment of the invention, the sdAb comprises a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71 . In a more preferred embodiment, the CDR3 region has as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71.
Accordingly, in a preferred embodiment of the invention, the sdAb comprises a CDR3 region from one of the identified preferred families, namely from any one of Family 8, 10, 13, 21 and 23, such as having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:24, SEQ ID NO: 59, SEQ ID NO:28, SEQ ID NQ:60, SEQ ID NO: 61 , SEQ ID NQ:20, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71. In a more preferred embodiment, the CDR3 region has as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 57, SEQ ID NO: 58 SEQ ID NO:24, SEQ ID NO: 59, SEQ ID NO:28, SEQ ID NQ:60, SEQ ID NO: 61 , SEQ ID NQ:20, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71.
The single-domain antibody defined by its CDR3 region can furthermore be defined as comprising a CDR1 region. The single-domain antibody of the invention may comprise a CDR3 as defined herein and further comprise a CDR1 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NQ:30, SEQ ID NO:42, SEQ ID NQ:50, SEQ ID NO:54, SEQ ID NQ:107, SEQ ID NO:111 , SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:147, SEQ ID NO:159, SEQ ID NO:163, SEQ ID NO:183, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:199, SEQ ID NO:211 , SEQ ID NO:215 and SEQ ID NO:239.
The single-domain antibody defined by its CDR3 region can furthermore be defined as comprising a CDR2 region. The single-domain antibody of the invention may comprise a CDR3 as defined herein and further comprise a CDR2 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:39, SEQ ID NO:51 , SEQ ID NO:136, SEQ ID NO:164, SEQ ID NO:172, SEQ ID NQ:180, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:192, SEQ ID NO:212 and SEQ ID NO:236.
The single-domain antibody defined by its CDR3 region can furthermore be defined as further comprising a CDR1 region and a CDR2 region. The single-domain antibody of the invention may comprise a CDR3 as defined herein and further comprise a CDR1 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NQ:30, SEQ ID NO:42, SEQ ID NQ:50, SEQ ID NO:54, SEQ ID NQ:107, SEQ ID NO:111 , SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:147, SEQ ID NO:159, SEQ ID NO:163, SEQ ID NO:183, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:199, SEQ ID NO:211 , SEQ ID NO:215 and SEQ ID NO:239, and further comprise a CDR2 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:39, SEQ ID NO:51 , SEQ ID NO:136, SEQ ID NO:164, SEQ ID NO:172, SEQ ID NQ:180, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:192, SEQ ID NO:212 and SEQ ID NO:236. In a preferred embodiment, the single-domain antibody is selected from the group comprising a. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:2, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 3, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 4; b. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:6, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 7, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 8; c. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 10, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 11 , and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 12; d. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 14, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 15, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:16; e. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 18, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 19, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 20;
f. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:22, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 23, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 24 g. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:26, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 27, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 28; h. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NQ:30, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 31 , and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 32; i. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:34, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 35, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 36; j. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:38, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 39, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 40; k. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:42, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 43, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 44; l. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:46, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 47, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 48; m. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NQ:50, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 51 , and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 52; n. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:54, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 55, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 56; o. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:83, a CDR2 region having at least 75% sequence identity to SEQ ID NO:84, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:85; p. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:87, a CDR2 region having at least 75% sequence identity to SEQ ID NO:88, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:89; q. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:91 , a CDR2 region having at least 75% sequence identity to SEQ ID NO:92, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:93;
r. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:95, a CDR2 region having at least 75% sequence identity to SEQ ID NO:96, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:97; s. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:99, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:100, and a CDR3 region having at least 75% sequence identity to SEQ ID NQ:101 ; t. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 103, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 104, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 105; u. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 107, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 108, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 109; v. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 111 , a CDR2 region having at least 75% sequence identity to SEQ ID NO: 112, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 113; w. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 115, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 116, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 117; x. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 119, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 120, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 121 ; y. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 123, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 124, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 125; z. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 127, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 128, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 129; aa. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:131 , a CDR2 region having at least 75% sequence identity to SEQ ID NO: 132, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 133; bb. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 135, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 136, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 137; cc. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 139, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:140, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:141 ;
dd. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 143, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 144, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 145; ee. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 147, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 148, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 149; ff. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:151 , a CDR2 region having at least 75% sequence identity to SEQ ID NO: 152, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 153; gg. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 155, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 156, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 157; hh. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 159, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:160, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:161 ; ii. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 163, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 164, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 165; jj. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 167, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 168, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 169; kk. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 171 , a CDR2 region having at least 75% sequence identity to SEQ ID NO: 172, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 173;
II. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 175, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 176, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 177; mm. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 179, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 180, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:181 ; nn. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 183, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 184, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 185; oo. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 187, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 188, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 189;
pp. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:191 , a CDR2 region having at least 75% sequence identity to SEQ ID NO: 192, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 193; qq. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 195, a CDR2 region having at least 75% sequence identity to SEQ ID NO: 196, and a CDR3 region having at least 75% sequence identity to SEQ ID NO: 197; rr. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO: 199, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:200, and a CDR3 region having at least 75% sequence identity to SEQ ID NQ:201 ; ss. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NQ:203, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:204, and a CDR3 region having at least 75% sequence identity to SEQ ID NQ:205; tt. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NQ:207, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:208, and a CDR3 region having at least 75% sequence identity to SEQ ID NQ:209; uu. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:211 , a CDR2 region having at least 75% sequence identity to SEQ ID NO:212, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:213; vv. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:215, a CDR2 region having at least 75% sequence identity to SEQ ID NO:216, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:217; ww. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:219, a CDR2 region having at least 75% sequence identity to SEQ ID NQ:220, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:221 ; xx. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:223, a CDR2 region having at least 75% sequence identity to SEQ ID NO:224, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:225; yy. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:227, a CDR2 region having at least 75% sequence identity to SEQ ID NO:228, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:229; zz. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:231 , a CDR2 region having at least 75% sequence identity to SEQ ID NO:232, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:233; aaa. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:235, a CDR2 region having at least 75% sequence identity to SEQ ID NO:236, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:237;
bbb. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:239, a CDR2 region having at least 75% sequence identity to SEQ ID NO:240, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:241 ; and ccc. a single-domain antibody comprising a CDR1 region having at least 75% sequence identity to SEQ ID NO:243, a CDR2 region having at least 75% sequence identity to SEQ ID NO:244, and a CDR3 region having at least 75% sequence identity to SEQ ID NO:245.
In a preferred embodiment, a single-domain antibody has at least 80% sequence identity, such as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96, 97, 98, or 99% sequence identity, to a sequence of the invention.
In a further aspect the invention is directed to a single-domain antibody having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242. In a preferred embodiment, a single-domain antibody has at least 80% sequence identity, such as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96, 97, 98, or 99% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242.
In a preferred embodiment, the single-domain antibody comprises, consists essentially of, or consists of a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, preferably SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242, more preferably SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53.
A further aspect of the invention is directed to a single-domain antibody comprising a CDR3 region having a binding affinity for the antigen microneme protein 2 (MIC2), such as a binding affinity for one or more of MIC2 from Eimeria tenella (EtMIC2), MIC2 from Eimeria maxima (EmMIC2), and MIC2 from Eimeria acervulina (EaMIC2). The single-domain antibody may comprise as CDR3 is as defined by the invention.
As can be seen in Figure 1 , when aligned, the three MIC2 variants are similar, but still display sequence variation. EmMIC2 and EaMIC2 are most similar (68.9% identity (86.5% similarity)), whereas EtMIC2 is both longer and more dissimilar when compared to the other two (41.9% identity (66.0% similarity) between EtMIC2 and EmMIC2 and 40.7% identity (66.3% similarity) between EtMIC2 and EaMIC2). Due to the presence of conserved regions between Eimeria spp. subspecies, an object of the invention is an sdAb which is cross-reactive, binding more than one MIC2 variant. Furthermore, given that there is diversity in the MIC2 sequence, a further object of the invention is an sdAb that only have specificity against single MIC2 variants. An immunization strategy where all three variants were used for co-immunization increased the likelihood of cross-reactive sdAbs.
In an embodiment of the invention, the single-domain antibody has a Kd for EaMIC2 of 10 nM or less. In a typical embodiment, the single-domain antibody has a Kd for EaMIC2 of 10 nM or less and has a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO: 59 of Family 10 and SEQ ID NO:28, SEQ ID NQ:60 and SEQ ID NO: 61 of Family 13. In a preferred embodiment, the single-domain has a Kd for EaMIC2 of 8 nM or less, such as 7, 6, 5, 4 3, 2, or 1 nM or less and the CDR3 region has at least 85% sequence identity or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity, to a sequence selected from the group
consisting of SEQ ID NO:24 and SEQ ID NO: 59 of Family 10 and SEQ ID NO:28, SEQ ID NO:60 and SEQ ID NO: 61 of Family 13
In a further embodiment, the single-domain antibody has a Kd for EmMIC2 of 10 nM or less. In a typical embodiment, the single-domain antibody has a Kd for for EmMIC2 of 10 nM or less and has at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 57 and SEQ ID NO: 58 of Family 8 and SEQ ID NQ:20, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO: 64 of Family 21. In a preferred embodiment, the singledomain has a Kd for EmMIC2 of 8 nM or less, such as 7, 6, 5, 4 3, 2, or 1 nM or less and the CDR3 region has at least 85% sequence identity or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 57 and SEQ ID NO: 58 of Family 8 and SEQ ID NQ:20, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO: 64 of Family 21 .
In a preferred aspect of the invention, the single-domain antibody has cross-reactivity by having a binding affinity to at least two of EaMIC2, EmMIC2, and EtMIC2 wherein it has a binding affinity with a Kd of 10 nM or less, at least one of EaMIC2, EmMIC2 and EtMIC2. In a prefered embodiment, the single-domain antibody has a cross-reactivity by binding to at least two of EaMIC2, EmMIC2, and EtMIC2 with a Kd of 10 nM or less, preferably having a cross-reactivity by binding to all three of EaMIC2, EmMIC2, and EtMIC2 with a Kd of 10 nM or less and wherein the CDR3 region has at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71 of Family 23. In a preferred embodiment, the single-domain antibody has a cross-reactivity by binding to at least two of EaMIC2, EmMIC2, and EtMIC2 with a Kd of 8 nM or less, such as 7, 6, 5, 4 3, 2, or 1 nM or less and the CDR3 region has at least 85% sequence identity or at least 90% sequence identity, such as at least 95% sequence identity, such as 100% sequence identity to a sequence selected from the group consisting of of SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71 of Family 23.
In an embodiment of the invention, the single-domain antibody has cross-reactivity in having a binding affinity to at least two of EaMIC2, EmMIC2, and EtMIC2 with a Kd of 10 nM or less to at least one of EaMIC2, EmMIC2, and EtMIC2 and further comprises a CDR1 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NQ:30, SEQ ID NO:42, SEQ ID NQ:50, SEQ ID NO:54, SEQ ID NQ:107, SEQ ID NO:111 , SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:147, SEQ ID NO:159, SEQ ID NO:163, SEQ ID NO:183, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:199, SEQ ID NO:211 , SEQ ID NO:215 and SEQ ID NO:239.
The single-domain antibody has cross-reactivity by having a binding affinity to at least two of EaMIC2, EmMIC2, and EtMIC2 and furthermore having a Kd of 10 nM or less to at least one of EaMIC2, EmMIC2, and EtMIC2 and may further comprise a CDR2 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:39, SEQ ID NO:51 , SEQ ID NO:136, SEQ ID NO:164, SEQ ID NO:172, SEQ ID NQ:180, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:192, SEQ ID NO:212 and SEQ ID NO:236.
The single-domain antibody has cross-reactivity by having a binding affinity to at least two of EaMIC2, EmMIC2, and EtMIC2 and having a Kd of 10 nM or less to at least one of EaMIC2, EmMIC2, and EtMIC2 and may further have at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, preferably SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242, more preferably SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53.
Interestingly, it was found that sdAbs targeting EtMIC2 were highly similar in sequence. Particularly interesting is the highly similar CDR3 region. Previously, it was noted that these sdAbs also displayed a similar TRF (time resolved fluorescence) signal in a DELFIA assay, which underlines the similarity. This means that sdAbs with high affinity to EtMIC2 in our hands are highly conserved and therefore subject to optimization. Based on thorough review of affinity and sequence variation, sdAbs were selected for homodimer construction (Table 1). Emphasis was put on proven high affinity, both during ranking and for purified protein, as well as sequence diversity.
Sources of single-domain antibodies having a binding affinity for the antigen microneme protein 2 (MIC2)
Single-domain antibodies of the invention can be obtained in a method comprising the use of camels or alpacas. In one embodiment, alpacas were immunized with MIC2 antigens. The animals were injected with antigen a total of 5 times over a 35-day period. From each animal, blood was then collected. RNA was prepared from the blood, pooled, and used for cDNA synthesis. From cDNA, sdAb genes could be PCR amplified before being cloned into a phagemid vector. In this way, core libraries containing approximately 1 .8x109 genes of the correct size were generated. These libraries were panned, using phage display, over solid-phase coated MIC2 antigen for a total of three rounds. Each round further accumulated a pool of sdAbs binding MIC2. Screening with ELISA showed colonies which panned against EmMIC2, EaMIC2, and/or EtMIC2.
Fusion Proteins
A fusion protein may comprise an sdAb of the invention bonded directly or through the intermediacy of a linker to another protein or polypeptide. As such, the second polypeptide, in one embodiment, is not an sdAb and may be any polypeptide.
In a preferred embodiment, a fusion protein comprises two or more sdAbs of the invention, such as 2, 3, 4, 5 or 6 sdAbs, preferably 2, 3, or 4, more typically 2 or 3, more preferably 2.
The fusion protein may be a homodimer or heterodimer of two or more, such as two sdAbs of the invention. A homodimer is defined, in one embodiment, such that the two sdAbs have CDR3 regions which have at least 75% sequence identity to each other. Accordingly, the fusion protein may be a homodimer selected from the group consisting of a. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:4 b. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:8;
c. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO: 12; d. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO: 16; e. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:20; f. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:24; g. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:28; h. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:32; i. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:36; j. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:40; k. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:44; l. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:48; m. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:52; n. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:56.
Preferably, the fusion protein may be a homodimer selected from the group consisting of a. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:20; b. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:24; c. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:28; d. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:36; e. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:40; f. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:48; and
g. a first sdAb linked to a second sdAb, each sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:56.
In a further embodiment, the fusion protein is a homodimer selected from the group consisting a. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , b. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:5 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:5, c. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:9 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:9, d. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 13 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:13, e. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 17 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:17, f. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 , g. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:25 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:25, h. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 29 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:29, i. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:33 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:33, j. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:37 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:37, k. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:41 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:41 , l. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:45 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:45, m. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:49 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:49, n. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:53 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:53, o. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:82 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:82, p. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:86 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:86,
q. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:90 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:90, r. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:94 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:94, s. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:98 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:98, t. a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:102 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 102, u. a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:106 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 106, v. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:110 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 110, w. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:114 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 114, x. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:118 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 118, y. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:122 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:122, z. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:126 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:126, aa. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 130 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 130, bb. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 134 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 134, cc. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 138 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 138, dd. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:142 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:142, ee. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:146 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:146, ff. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 150 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 150, gg. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 154 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 154, hh. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 158 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 158, ii. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:162 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:162,
jj. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:166 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:166, kk. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 170 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 170,
II. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 174 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 174, mm. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 178 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:178, nn. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 182 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 182, oo. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 186 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 186, pp. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 190 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 190, qq. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 194 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 194, rr. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 198 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 198, ss. a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:202 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:202, tt. a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:206 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:206, uu. a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:210 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:210, vv. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:214 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:214, ww. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:218 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:218, xx. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:222 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:222, yy. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:226 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:226, zz. a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:230 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:230,
aaa. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:234 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:234, bbb. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:238 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:238, and ccc. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:242 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:242.
Preferably, the fusion protein is a homodimer selected from the group consisting of a. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 17 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 17, b. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 , c. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:25 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:25, d. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:33 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:33, e. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:37 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:37, f. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:45 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:45, and g. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:53 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:53.
A fusion protein may alternatively be defined, in another embodiment, as a heterodimer comprising two or more sdAbs, such as two sdAbs, of the invention. In such an embodiment, the fusion protein may be a heterodimer selected from the group consisting of a. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:4 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; b. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:8 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to
a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; c. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO: 12 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; d. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO: 16 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; e. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:20 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; f. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:24 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; g. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:28 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; h. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:32 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12,
SEQ ID N0:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:40,
SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; i. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID
NO:36 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NQ:40,
SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; j. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:40 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; k. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:44 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; l. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:48 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:52 and SEQ ID NO:56; m. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:52 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48 and SEQ ID NO:56; and n. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:56 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48 and SEQ ID NO:52.
In a preferred embodiment of a heterodimer, the fusion protein is a heterodimer selected from the group consisting of a. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:20 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; b. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:24 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; c. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:28 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; d. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:36 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56; e. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NQ:40 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56;
f. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:48 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:52 and SEQ ID NO:56 ; and g. a first sdAb comprising a CDR3 region having at least 75% sequence identity to SEQ ID NO:56 linked to a second sdAb comprising a CDR3 region having at least 75% sequence identity to a single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48 and SEQ ID NO:52.
Interestingly, fusion proteins that bind EtMIC2 strongly also show binding to EmMIC2. Since EtMIC2 on sequence level is the most divergent of the three MIC2 molecules, this could indicate that fusion proteins binding to EtMIC2 are interacting with a highly conserved binding site. As determined by BLI measurements, all fusion proteins of the invention have demonstrated binding affinities and having and a Kd of less than 10 nM, and less than 1 nM. Of particular interest is the embodiment where the fusion protein is selected from fusion proteins which display cross-binding to both EtMIC2 and EmMIC2, such as SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75.
In a further embodiment, the fusion protein is a heterodimer selected from the group consisting of a. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; b. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:5 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ
ID N0:9, SEQ ID N0:13, SEQ ID N0:17, SEQ ID N0:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID N0:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID N0:114, SEQ ID N0:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; c. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:9 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; d. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:13 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, e; e. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 17 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242;
f. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; g. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:25 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; h. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 29 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; i. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:33 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ ID
N0:5, SEQ ID N0:9, SEQ ID N0:13, SEQ ID N0:17, SEQ ID N0:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:37, SEQ ID N0:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID N0:114, SEQ ID N0:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; j. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:37 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; k. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:41 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; l. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:45 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:49, SEQ ID NO:53, SEQ ID
NO:82, SEQ ID NO:86, SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:110, SEQ ID N0:114, SEQ ID N0:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; m. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:49 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; n. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:53 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45 and SEQ ID NO:49, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; o. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:82 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126,
SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; p. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:86 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; q. a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:90 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; r. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:94 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170,
SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; s. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:98 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; t. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 102 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; u. a single-domain antibody having at least 75% sequence identity to SEQ ID
NO: 106 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25,
SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98,
SEQ ID NQ:102, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID
NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID
NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; v. a single-domain antibody having at least 75% sequence identity to SEQ ID
NQ:110 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25,
SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98,
SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID
NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; w. a single-domain antibody having at least 75% sequence identity to SEQ ID
NO: 114 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25,
SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98,
SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:118, SEQ ID NO:122, SEQ ID
NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; x. a single-domain antibody having at least 75% sequence identity to SEQ ID
NO:118 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25,
SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98,
SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID
NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242;
y. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 122 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; z. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 126 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; aa. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 130 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; bb. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 134 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:
1 , SEQ ID N0:5, SEQ ID N0:9, SEQ ID N0:13, SEQ ID N0:17, SEQ ID N0:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID N0:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID N0:114, SEQ ID N0:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; cc. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 138 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; dd. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 142 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; ee. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 146 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NQ:110, SEQ ID N0:114, SEQ ID N0:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; ff. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 150 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; gg. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 154 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; hh. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 158 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID
NO:122, SEQ ID NO:126, SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; ii. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 162 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; jj. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 166 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; kk. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 170 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID
NO:166, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242;
II. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 174 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; mm. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 178 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; nn. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 182 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID
NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NO:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; oo. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 186 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID N0:5, SEQ ID N0:9, SEQ ID N0:13, SEQ ID N0:17, SEQ ID N0:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID N0:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID N0:114, SEQ ID N0:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; pp. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 190 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; qq. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 194 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242;
rr. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 198 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25,
SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98,
SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID
NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; ss. a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:202 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25,
SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98,
SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID
NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; tt. a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:206 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25,
SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98,
SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID
NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; uu. a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:210 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:
1 , SEQ ID N0:5, SEQ ID N0:9, SEQ ID N0:13, SEQ ID N0:17, SEQ ID N0:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID N0:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID N0:114, SEQ ID N0:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; vv. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:214 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; ww. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:218 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; xx. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:222 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NQ:110, SEQ ID N0:114, SEQ ID N0:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; yy. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:226 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; zz. a single-domain antibody having at least 75% sequence identity to SEQ ID NQ:230 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; aaa. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:234 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID
NO:122, SEQ ID NO:126, SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:238 and SEQ ID NO:242; bbb. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:238 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25,
SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98,
SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234 and SEQ ID NO:242; and ccc. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:242 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25,
SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98,
SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234 and SEQ ID NO:238.
A preferred heterodimer is one in which the fusion protein is selected from the group consisting of a. a single-domain antibody having at least 75% sequence identity to SEQ ID NO: 17 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53; b. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:21 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53;
c. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:25 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53; d. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:33 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53; e. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:37 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:45 and SEQ ID NO:53; f. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:45 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37 and SEQ ID NO:53; and g. a single-domain antibody having at least 75% sequence identity to SEQ ID NO:53 linked to a single-domain antibody having at least 75% sequence identity to SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37 and SEQ ID NO:45.
The fusion protein of the invention typically comprises two single-domain antibodies covalently bound to each other, preferably at the C-terminus of the first single-domain antibody to the N-terminus of the second single-domain antibody. The C-terminus of the first single-domain antibody may be directly covalently bound to the N-terminus of the second single-domain antibody or by means of a linker. Preferably the N-terminus of the first single-domain antibody is bound to the C-terminus of the second single-domain antibody by means of a linker. The linker is preferably stable to gastric conditions. The linker is typically from 1 to 20 nm in length, preferably from 3 to 15 nm, such as 3 to 12 nm.
In preferred embodiments of the invention, the linker is a stable linker and confers stability to the protein of the invention. Thus, upon exposure as described herein below, said linker is not degraded and hence is capable of maintaining a link between said first and said second sdAb of the present invention. Preferably, the protein is stable in the gastrointestinal tract and/or respiratory system of a subject, i.e., the linker is capable of remaining intact in the gastrointestinal tract and/or respiratory system of a subject. In one embodiment, the protein is acid stable and/or protease stable. The protein may be stable in the presence of any acids, such as bile acid and/or citric acid. The fusion protein is also preferably stable, when in a composition or formulation of the invention, in the presence of any proteases, such as pepsin and/or trypsin. In one embodiment, the fusion protein, when in a composition or formulation of the invention, is stable in the presence of gall, such as 50%, 10%, or 2% gall for 1 hour. In another embodiment, the protein is stable in the presence of a bile salt, such as 0.2 mM, 1 mM, and 10 mM sodium deoxycholate (NaDeox) for 1 hour.
In one embodiment of the present invention, the protein is temperature stable, such as temperature stable until 70 °C. This indicates high protein stability. In another embodiment, the
protein is pH stable. Unexpectedly, the inventors found that, at pH 3- 4, approx. 70-90% of the protein remained stable, thus said protein is able to pass through the gastrointestinal tract as at least a dimeric fusion protein and retain its activity.
In an embodiment of the invention, the first and second scAb are linked via a linker polypeptide comprising 5 to 50 amino acid residues, such as 10 to 40 amino acid residues, such as 10 to 30 amino acid residues.
In a preferred embodiment, the fusion protein comprises two single-domain antibodies covalently bound to each other, wherein the C terminus of the first single-domain antibody is bound to the N terminus of the second single-domain antibody by means of a linker, wherein the linker is a polypetide. In a preferred embodiment, the linker comprises or consists of glycine and serine residues. In one embodiment, the linker is a GS linker, such as a GS linker of the structure (GxS)n, where x may be a number between 1 to 10, preferably 3 to 8, such as 2 to 6 or 2 to 5, and n refers to a number of repeats of the GxS sequence, where n may be between 1 to 10, preferably 2 to 5. In one embodiment, the linker comprises (GGGGS)n where n is 1 to 10, preferably 2 to 5, such as 2, 3, 4 or 5.
In suitable embodiments, the GS linker is a GGGGS linker, a GGGGSGGGGS linker, a GGGGSGGGGSGGGGS linker, a GGGGSGGGGSGGGGSGGGGS linker, a GGGGSGGGGSGGGGSGGGGSGGGGS linker, or a GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker. In a preferred embodiment, the linker is (GGGGS)3. The length of the (GGGGS)3 linker was calculated to be about 5.7 nm.
A further aspect of the invention is directed to a fusion protein selected from the group consisting of: a. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:72; b. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:73; c. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:74; d. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95%
sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:75; e. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:76; f. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:77; g. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:78; h. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:79; i. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NQ:80; and j. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:81.
In one aspect of the invention, the fusion protein comprises two single-domain antibodies of the invention and has a cross-reactivity by having binding affinity to at least two of EaMIC2, EmMIC2, and EtMIC2 and having a Kd of less than 10 nM to at least one of EaMIC2, EmMIC2, and EtMIC2, such as less than 8 nM, such as less than 7, 6, 5, 4, 3, 2 or 1 nM, preferably a crossreactivity by having a binding affinity to each of EaMIC2, EmMIC2 and EtMIC2.
In a preferred embodiment, the fusion protein of the invention retains binding affinity after thermal treatment such that the fusion protein retains at least 60%, such as at least 65%, such as at least 75% of its binding affinity measured prior to thermal treatment, as performed in Example 7.
Protein Granules
The sdAb are typically formulated for use for adminstration to an animal. A further aspect of the invention is directed to a composition comprising an sdAb of the invention. Similarly, the invention is directed to a composition comprising the fusion protein of the invention. The composition typically further comprises a preservative, filler, and/or carrier material. The composition may be a liquid composition, solid composition, solution, dispersion, paste, powder, granule, granulate, coated granulate, tablet, cake, crystal, crystal slurry, gel or pellet.
The granule suitably comprises:
(a) a core comprising the single-domain antibody or the fusion protein of the invention and optionally,
(b) a coating consisting of one or more layer(s) surrounding the core.
In one embodiment, the granule comprises:
(a) a core, and
(b) a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises the single-domain antibody or the fusion protein of the invention
The present invention also relates to granules/particles comprising a single-domain antibody of the invention. In an embodiment, the granule comprises a core, and optionally one or more coatings (outer layers) surrounding the core. The core may have a diameter, measured as equivalent spherical diameter (volume based average particle size), of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm. The core diameter, measured as equivalent spherical diameter, can be determined using laser diffraction, such as using a Malvern Mastersizer and/or the method described under ISO13320 (2020). In an embodiment, the core comprises a single-domain antibody or fusion protein of the present invention.
The core may include additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances. The core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate. The core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend. The core may include an inert particle with the single-domain antibody absorbed into it, or applied onto the surface, e.g., by fluid bed coating. The core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250- 1200 pm.
The core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule. The optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). The coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%. The amount may be at most 100%, 70%, 50%, 40% or 30%. The
coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In some embodiments, the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm. The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit has few or no uncoated areas. The layer or coating should, in particular, be homogeneous in thickness.
The coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
A salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight. The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm. The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular, having a solubility at least 0.1 g in 100 g of water at 20 °C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water. The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.
The salt in the coating may have a constant humidity at 20 °C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in WO 00/01793 or WO 2006/034710.
Specific examples of suitable salts are NaCI (CH20 °c=76%), Na2CO3 (CH20 °c=92%), NaNO3 (CH2O"C=73%), Na2HPO4 (CH2o"c=95%), Na3PO4 (CH25°c=92%), NH4CI (CH2o"c= 79.5%), (NH4)2HPO4 (CH2O "C = 93,0%), NH4H2PO4 (CH20 -c = 93.1 %), (NH4)2SO4 (CH20 °c=81 .1%), KOI (CH2O"C=85%), K2HP04 (CH2O"C=92%), KH2P04 (CH2O"C=96.5%), KNO3 (CH2O"C=93.5%), Na2SO4 (CH20 »c=93%), K2SO4 (CH20 »c=98%), KHSO4 (CH2O »C=86%), MgSO4 (CH20 »c=90%), ZnSO4 (CH2O°C=9O%) and sodium citrate (CH25°c=86%). Other examples include NaH3PO4, (NH4)H3PO4, CuSO4, Mg(NO3)2 and magnesium acetate.
The salt may be in anhydrous form, or it may be a hydrated salt, i.e., a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfate heptahydrate (MgSO4 ?H2O), zinc sulfate heptahydrate (ZnSO4 ?H2O), sodium phosphate dibasic heptahydrate (Na2HPO4 ?H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.
Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed.
The coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
The granule may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme granules with multiple coatings are described in WO 93/07263 and WO 97/23606.
The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
Methods for preparing the core can be found in the Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Vol. 1 ; 1980; Elsevier. Preparation methods include known feed and granule formulation technologies, e.g.,
(a) Spray dried products, wherein a liquid single-domain antibody-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form a single-domain antibody-containing particulate material. Very small particles can be produced this way (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; Vol. 71 , pages 140-142; Marcel Dekker).
(b) Layered products, wherein the single-domain antibody is coated as a layer around a pre-formed inert core particle, wherein a single-domain antibody-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the single-domain antibody-containing solution adheres to the core particles and dries up to leave a layer of dry single-domain antibody on the surface of the core particle. Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found. This type of product is described in, e.g., WO 97/23606.
(c) Absorbed core particles, wherein rather than coating the single-domain antibody as a layer around the core, the single-domain antibody is absorbed onto and/or into the surface of the core. Such a process is described in WO 97/39116.
(d) Extrusion or pelletized products, wherein a single-domain antibody-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the single-domain antibody paste, which is harmful to the single-domain antibody (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; Vol. 71 ; pages 140-142; Marcel Dekker).
(e) Prilled products, wherein a single-domain antibody-containing powder is suspended in molten wax and the suspension is sprayed, e.g., through a rotating disk atomizer, into a cooling chamber where the droplets quickly solidify (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; Vol. 71 ; pages 140-142; Marcel Dekker). The product obtained is one wherein the single-domain antibody is uniformly distributed throughout an inert material instead of being concentrated on its surface. US 4,016,040 and US 4,713,245 describe this technique.
(f) Mixer granulation products, wherein a single-domain antibody-containing liquid is added to a dry powder composition of conventional granulating components. The liquid and the powder in a suitable proportion are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the single-domain antibody. Such a process is described in US 4,106,991 , EP 170360, EP 304332, EP 304331 , WO 90/09440 and WO 90/09428. In a particular aspect of this process, various high-shear mixers can be used as granulators. Granulates consisting of single-domain antibody, fillers and binders etc. are mixed with cellulose fibers to reinforce the particles to produce a so-called T-granulate. Reinforced particles, are more robust, and release less enzymatic dust.
(g) Size reduction, wherein the cores are produced by milling or crushing of larger particles, pellets, tablets, briquettes etc. containing the single-domain antibody. The wanted core particle fraction is obtained by sieving the milled or crushed product. Over and undersized particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.
(h) Fluid bed granulation. Fluid bed granulation involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them to form a granule.
(i) The cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or enzyme industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90 °C. For some singledomain antibodies, it is important the cores comprising the single-domain antibody contain a low amount of water before coating with the salt. If water sensitive single-domain antibodies are coated with a salt before excessive water is removed, the excessive water will be trapped within the core and may affect the activity of the single-domain antibody negatively. After drying, the cores preferably contain 0.1-10% w/w water.
Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and US 4,661 ,452 and may optionally be coated by methods known in the art.
The granulate may further comprise other actives, such as enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase. The one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, betaglucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta-xylosidase or any combination thereof. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D.
Liquid Formulations
The present invention also relates to liquid compositions comprising a single-domain antibody of the invention or the fusion protein of the invention. The composition may comprise a stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid). The liquid composition comprising the single-domain antibody or the fusion protein may comprise a stabilizer, e.g., a polyol such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid). The liquid composition may further comprise a filler or carrier material. The liquid composition may further comprise a preservative.
In some embodiments, filler(s) or carrier material(s) are included to increase the volume of such compositions. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like. Suitable filler or carrier
materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials.
In an aspect, the liquid formulation comprises 20-80% w/w of polyol. In one embodiment, the liquid formulation comprises 0.001-2% w/w preservative. In another embodiment, the invention relates to liquid formulations comprising:
(A) 0.001-25% w/w of a single-domain antibody or fusion protein of the present invention;
(B) 20-80% w/w of polyol;
(C) optionally 0.001-2% w/w preservative; and
(D) water.
In another embodiment, the invention relates to liquid formulations comprising:
(A) 0.001-25% w/w of a single-domain antibody or fusion protein of the present invention;
(B) 0.001-2% w/w preservative;
(C) optionally 20-80% w/w of polyol; and
(D) water.
In another embodiment, the liquid formulation comprises one or more formulating agents, such as a formulating agent selected from the group consisting of polyol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment, the polyols is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600, more preferably selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.
In another embodiment, the liquid formulation comprises 20-80% polyol (/.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol. In one embodiment, the liquid formulation comprises 20-80% polyol, e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40%-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600. In one embodiment, the liquid formulation comprises
20-80% polyol (/.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG).
In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02-1.5% w/w preservative, e.g., 0.05-1% w/w preservative or 0.1-0.5% w/w preservative. In one embodiment, the liquid formulation comprises 0.001-2% w/w preservative (/.e., total amount of preservative), e.g., 0.02- 1.5% w/w preservative, 0.05-1 % w/w preservative, or 0.1 -0.5% w/w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.
In another embodiment, the liquid formulation further comprises one or more enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase. The one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, betagalactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha- mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta- xylosidase or any combination thereof.
In an embodiment, the liquid composition of the invention comprising a single-domain antibody or fusion protein is in the form of an aqueous solution, in particular a solution which is suitable to be added to drinking water for an animal such as a monogastric animal. It is thus contemplated that the single-domain antibodies or fusion proteins may be administered via drinking water rather than in the form of e.g. an animal feed or animal feed additive.
Animal feed and animal feed additives
A further aspect of the invention is directed to animal feed and animal feed additives. The invention is directed to a feed additive comprising an sdAb of the invention or the fusion protein of the invention and to a feed comprising said feed additive or an sdAb of the invention or the fusion protein of the invention. The present invention relates to animal feed compositions and animal feed additives comprising an sdAb of the invention or a fused protein of the invention, a composition of the invention, a granule of the invention, or a liquid formulation of the invention. Typically, according to the invention, the feed or feed additive is for a monogastric animal, typically for swine, piglet, growing pig, sow, poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick, cat, dog, or horse. Preferably, the animal feed or animal feed additive is for a monogastric animal from the group consisting of poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick.
Even more preferably, the animal feed or animal feed additive is for a monogastric animal selected from the group consisting of chicken, broiler, layer, and chick. In a preferred embodiment, the animal feed or animal feed additive is for use in poultry. In a further aspect of the invention, the animal feed or animal feed additive is for a household pet, such as a dog or cat.
Animal feed compositions or diets have a relatively high content of protein. Poultry and pig diets can be characterised as indicated in Table B of WO 01/58275, columns 2-3. Fish diets can be characterised as indicated in column 4 of this Table B. Furthermore, such fish diets usually have a crude fat content of 200-310 g/kg.
An animal feed composition according to the invention has a crude protein content of 50- 800 g/kg, and furthermore comprises an sdAb or fused protein of the invention.
Furthermore, or in the alternative (to the crude protein content indicated above), the animal feed composition of the invention has a content of metabolisable energy of 10-30 MJ/kg; and/or a content of calcium of 0.1-200 g/kg; and/or a content of available phosphorus of 0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or a content of methionine plus cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50 g/kg.
In particular embodiments, the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO 01/58275 (R. 2-5). Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25, i.e. Crude protein (g/kg)= N (g/kg) x 6.25. The nitrogen content is determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC). Metabolisable energy can be calculated on the basis of the NRC publication Nutrient requirements in swine, ninth revised edition 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, D.C., pp. 2-6, and the European Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN 90- 71463-12-5. The dietary content of calcium, available phosphorus and amino acids in complete animal diets is calculated on the basis of feed tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.
In a particular embodiment, the animal feed composition of the invention contains at least one vegetable protein. The animal feed may comprise vegetable proteins. In particular embodiments, the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w). Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae (Leguminosae), Brassicaceae, Amaranthaceae, and Poaceae, such as soybean meal, lupin meal, rapeseed meal, and combinations thereof.
In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean. In another particular embodiment, the vegetable protein source is material from one or more plants of the family Amaranthaceae, e.g. beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rapeseed, crambe and cabbage. In another particular embodiment, soybean is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.
The animal feed composition of the invention may also contain animal protein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal, typically in an amount of 0-25%. The animal feed composition of the invention may also comprise Dried Distillers Grains with Solubles (DDGS), typically in amounts of 0-30%.
The animal feed composition of the invention may also contain insect protein, such as protein from mealworm, housefly or black soldier fly larvae, typically in meal form. Insect meal may replace fishmeal entirely or in part, and thus may constitute 0-10% of the total feed.
In still further particular embodiments, the animal feed composition of the invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or 0-20% whey.
Animal diets can e.g. be manufactured as mash feed (non-pelleted) or pelleted feed. Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question, an sdAb or fused protein of the invention can be added as solid or liquid enzyme formulations. For example, for mash feed a solid or liquid an sdAb or fused protein formulation of the invention may be added before or during the ingredient mixing step. For pelleted feed the (liquid or solid) alpha-, an sdAb or fused protein preparation may also be added before or during the feed ingredient step. Typically, an sdAb or fused protein preparation optionally comprises a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets. The sdAb or fused protein may also be incorporated in a feed additive or premix.
In an embodiment, the animal feed or animal feed additive comprises one or more enzymes. In an embodiment, the animal feed comprises one or more microbes. In an embodiment, the animal feed comprises one or more vitamins. In an embodiment, the animal feed comprises one or more minerals. In an embodiment, the animal feed comprises one or more amino acids. In an embodiment, the animal feed comprises one or more other feed ingredients.
In a further embodiment, the animal feed or animal feed additive comprises the polypeptide of the invention, one or more formulating agents and one or more components selected from the list consisting of: one or more enzymes; one or more microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.
In an embodiment, the animal feed additive comprises one or more formulating agents. In an embodiment, the animal feed additive comprises one or more probiotics. In an embodiment, the animal feed additive comprises one or more vitamins. In an embodiment, the animal feed additive comprises one or more minerals. In an embodiment, the animal feed additive comprises one or more amino acids. In an embodiment, the animal feed additive comprises one or more prebiotics. In an embodiment, the animal feed additive comprises one or more organic acids. In an embodiment, the animal feed additive comprises one or more phytogenies.
Methods of Improving Animal Performance or Health
The invention further relates to a method of improving one or more performance parameters of an animal, comprising administering to an animal the animal feed additive of the invention.
In one aspect, an animal feed is prepared from the animal feed additive, granule or liquid formulation as described herein and administered to the animal. The invention further relates to a method of improving one or more performance parameters of an animal, comprising administering to one or more animals an animal feed or pelleted animal feed comprising the sdAb or fused protein of the invention. An aspect of the invention is directed to a method of increasing the food conversion ration (FCR) or body weight gain in animal comprising administering to said animal a feed or feed additive comprising a single-domain antibody or fusion protein of the invention.
In one embodiment, ‘improving the performance of an animal’ means that there is an increase in body weight gain. In another embodiment, ‘improving the performance of an animal’ means that there is an improved feed conversion ratio. In a further embodiment, ‘improving the performance of an animal’ means that there is an increased feed efficiency. In a further embodiment, ‘improving the performance of an animal’ means that there is an increase in body weight gain and/or an improved feed conversion ratio and/or an increased feed efficiency.
The invention, in one aspect, is directed to supplementing an animal feed with an sdAb or fused protein of the invention to improve a parameter in an animal, typically for swine, piglet, growing pig, sow, poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick, cat, dog, or horse. Preferably, the parameter is improved in a monogastric animal, such as selected from the group consisting of poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick. Even more preferably, the parameter is improved in an animal selected from the group consisting of chicken, broiler, layer, and chick. In a further aspect of the invention, the parameter is in a is for a household pet, such as a dog or cat.
The invention, in one aspect, is directed to supplementing an animal feed with an sdAb or fused protein of the invention to improve a parameter in an animal such as a chicken, such as a broiler chicken wherein the parameter is selected from the group consisting of
a) growth performance such as weight gain, feed intake, and/or food conversion ratio (FCR); b) macroscopic and histological lesions, c) expression of anti-inflammatory cytokines such as IL10 and IL8; and/or d) carotenoids content in the blood.
The invention, in a further aspect, is directed to supplementing an animal feed with an sdAb or fused protein of the invention to improve a parameter in animal such as a chicken, such as a broiler chicken which is susceptible to infection by Eimeria and Clostridium perfringes, wherein the parameter is selected from the group consisting of a) growth performance such as weight gain, feed intake, and/or food conversion ratio (FCR); b) macroscopic and histological lesions, c) expression of anti-inflammatory cytokines such as IL10 and IL8; and/or d) carotenoids content in the blood.
The invention is furthermore directed to a method of treating, preventing or improving infections, such as Eimeria and Clostridium perfringens infections, or other parasitic infections, in a monogastric animal comprising administering to the animal a composition, an animal feed or an animal feed additive comprising an sdAb or fused protein of the invention.
For the Eimeria spp. parasite species to be successful, they need to both move a distance in the gut and invade a chicken intestinal cell. If either, or both, of these actions is inhibited, the parasite lifecycle will be inhibited. Accordingly, one embodiment of the invention relates to sdAbs or fusion proteins of the invention which limit mobility of the parasite and/or reduces invasion of intestinal cells. If the parasite lifecycle is inhibited, the risk of coccidiosis is lowered. Accordingly, one embodiment of the invention relates to sdAbs or fusion proteins of the invention which limit mobility of the parasite and/or reduces invasion of intestinal cells and/or inhibits the lifecycle of the parasite. Among the different species of Eimeria spp., three are the most important to address for poultry coccidiosis, namely Eimeria tenella, Eimeria maxima, and Eimeria acervulina. Accordingly, one embodiment of the invention relates to sdAbs or fusion proteins of the invention which limit mobility of any one of Eimeria tenella, Eimeria maxima, and Eimeria acervulina and/or reduces invasion of intestinal cells and/or inhibits the lifecycle of any one of Eimeria tenella, Eimeria maxima, and Eimeria acervulina. ‘Microneme proteins’ are essential for host invasion and movement. Accordingly, a further aspect of the invention is directed to to sdAbs or fusion proteins of the invention which binding to Microneme proteins, preferably Microneme protein 2 (MIC2).
In the present invention, the parasitic infection is typically in an animal selected from the group consisting of swine, piglet, growing pig, sow, poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick, cat, dog, horse. Preferably, the parasitic infection is in an animal selected from the group consisting of poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick. Even more preferably, the parasitic infection is typcially in an animal selected from the group consisting of chicken, broiler,
layer, and chick. In a further aspect of the invention, the parameter is in a is for a household pet, such as a dog or cat.
An aspect of the invention is directed supplementing an animal feed with a single-domain antibody or fusion protein of the invention, to treat, prevent or improve/reduce an infection, such as Eimeria and Clostridium perfringens infections, in a monogastric animal, compared to an animal feed without the supplementation. A further aspect of the invention is directed to a method of treating or preventing coccidiosis in poultry comprising the use of a granule, composition, feed additive or feed of the invention. Similarly, the invention is directed to a use of a fusion protein or an sdAb for the preparation of a medicament for the prevention or treatment of coccidiosis in poultry. Similarly, a further aspect is directed to a method of preventing or treating Eimeria or coccidiosis in poultry comprising administering a feed or additive of the invention. The invention is furthermore directed to a method of reducing parasites or reducing the risk of infections in an animal, such as an animal selected from the group consisting of swine, piglet, growing pig, sow, poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick, and horse. A further embodiment is directed to a method of reducing parasites or reducing the risk of infections in poultry.
A further aspect is directed to a method of reducing parasites or reducing the risk of infections in a pet, such as but not limited to a dog, cat, bird, or rabbit.
In the present invention, the sdAb or fused protein of the invention may be fed to the animal from birth until slaughter. Preferably, the sdAb or fusion protein of the invention is fed to the animal on a daily basis from birth until slaughter. More preferably, the sdAb or fusion protein of the invention is fed to the animal on a daily basis for at least 10 days, such as at least 15 days or at least 20 days (where the days can be continuous or non-continuous) during the life span of the animal. Further preferably, the sdAb or fusion protein of the invention is fed to the animal for 10-20 days followed by a non-treatment period of 5-10 days, and this cycle is repeated during the life span of the animal. In the present invention, the sdAb or fusion protein of the invention may be fed to broilers for the first 49 days after hatching. Preferably, the sdAb or fusion protein of the invention is fed to broilers for the first 36 days after hatching. More preferably, the sdAb or fusion protein of the invention is fed to broilers on days 22 to 36 after hatching. Further preferably, the sdAb or fusion protein of the invention is fed to broilers during the pre-starter (days 1-7) period. Further preferably, the sdAb or fusion protein of the invention is fed to broilers during the starter (days 8-22) period. Further preferably, the sdAb or fusion protein of the invention is fed to broilers during the pre-starter (days 1- 7) and starter (days 8-22) period. In the present invention, the sdAb or fusion protein of the invention may be fed to layers during the life span of the animal. Preferably, the sdAb or fusion protein of the invention is fed to layers for 76 weeks from hatching. More preferably, the sdAb or fusion protein of the invention is fed to layers during the laying period, (from ca. week 18). Further preferably, the sdAb or fusion protein of the invention is fed to layers during the laying period but withheld during the forced molting period.
In the present invention, the sdAb or fusion protein of the invention may be fed to turkeys during life span of the animal. Preferably, the sdAb or fusion protein of the invention is fed to turkeys for 24 weeks from hatching. More 30 preferably, the sdAb or fusion protein of the invention is fed to turkeys for the first 16 weeks from hatching (for hens) and for the first 20 weeks for hatching (for toms). In the present invention, the sdAb or fusion protein of the invention may be fed to swine during life span of the animal. Preferably, the sdAb or fusion protein of the invention is fed to swine for 27 weeks from birth. More preferably, the sdAb or fusion protein of the invention is fed to piglets from birth to weaning (at 4 weeks). Further preferably, the sdAb or fusion protein of the invention is fed to piglets for the first 6 weeks from birth (4 weeks of lactation and 2 weeks post-weaning). Further preferably, the sdAb or fusion protein of the invention is fed to weaning piglets during the pre-starter (days 1-14 after weaning) Further preferably, the sdAb or fusion protein of the invention is fed to weaning piglets during the starter (days 15-42 after weaning) period. Further preferably, the sdAb or fusion protein of the invention is fed to weaning piglets during the pre-starter (days 1-14 after weaning) and starter (days 15-42 after weaning) period. Further preferably, the sdAb or fusion protein of the invention is fed to swine during the grower/fattening period (week 10 to ca. week 27 after birth).
A further aspect of the invention is directed to an sdAb or fusion protein comprising an sdAb having a binding affinity for the antigen microneme protein 2 as defined by the invention for the preparation of an animal feed additive or animal feed.
The sdAb of the invention or the fusion protein of the invention may also be formulated as animal feed or animal feed additive for treating, preventing, or reducing the spread of Eimeria or Clostridium perfringens infections, of an animal.
As noted above, it is contemplated that the single-domain antibodies or fusion proteins may be administered not only as part of an animal feed or animal feed additive, but alternatively or additionally via drinking water. One aspect of the invention thus relates to method for treating, preventing, or reducing the spread of Eimeria or Clostridium perfringens infections in an animal such as a monogastric animal, the method comprising administering to the animal drinking water comprising a single-domain antibody or fusion protein of the invention.
One aspect of the invention is directed to use of a composition, an animal feed, or an animal feed additive for treating, preventing, or reducing the spread of Eimeria and Clostridium perfringens infections of a monogastric animal wherein the composition, the animal feed or the animal feed additive comprises the fusion protein or sdAb of the invention. Similarly, the invention is directed to a method of treating, preventing or or reducing the spread of Eimeria and Clostridium perfringens infections, of a monogastric animal comprising administering to the animal a composition, an animal feed or an animal feed additive comprising the fusion protein or sdAb of the invention. Typically, the animal is a bird, more typically poultry.
An aspect of the invention is directed to use of an sdAb or fusion protein comprising an sdAb having a binding affinity for the antigen microneme protein 2 (MIC2) in an animal feed or animal feed additive for the prevention or treatment of a parasitic infection, wherein the sdAb is selected from the group consisting of:
(a) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 1;
(b) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 5;
(c) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 9;
(d) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 13;
(e) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 17;
(f) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 21 ;
(g) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 25;
(h) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 29;
(i) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 33;
(j) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 37;
(k) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 41 ;
(l) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 45;
(m) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 49;
(n) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 53;
(o) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 82;
(p) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 86;
(q) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 90;
(r) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 94;
(s) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 98;
(t) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 102;
(u) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 106;
(v) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 110;
(w) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 114;
(x) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 118;
(y) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 122;
(z) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 126;
(aa) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 130;
(ab) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 134;
(ac) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 138;
(ad) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 142;
(ae) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 146;
(af) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 150;
(ag) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 154;
(ah) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 158;
(ai) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 162;
(aj) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 166;
(ak) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 170;
(al) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 174;
(am) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 178;
(an) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 182;
(ao) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 186;
(ap) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 190;
(aq) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 194;
(ar) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 198;
(as) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 202;
(at) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 206;
(au) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 210;
(av) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 214;
(aw) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 218;
(ax) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 222;
(ay) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 226;
(az) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 230;
(ba) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 234;
(bb) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 238;
(be) a single-domain antibody having at least 60% sequence identity to SEQ ID NO: 242;
(bd) a single-domain antibody encoded by a polynucleotide having at least 80% sequence identity to the coding sequence of any one of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; preferably SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, more preferably SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53;
(be) a single-domain antibody derived from any one of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID
NO:33, SEQ ID NO:37, SEQ ID N0:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID N0:114, SEQ ID N0:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242; preferably SEQ ID N0:5, SEQ ID N0:9, SEQ ID N0:17, SEQ ID N0:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID N0:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, more preferably SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53 by having 1-20 alterations (e.g., substitutions, deletions and/or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 alterations, in particular substitutions; and
(bf) a single-domain antibody derived from the single-domain antibody of (a), (b), (c), (d), (e), (f). (g). (h), (i), 0), (k), (I), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), (x), (y), (z), (aa), (ab), (ac), (ad), (ae), (af), (ag), (ah), (ai), (aj), (ak), (al), (am), (an), (ao), (ap), (aq), (ar), (as), (at), (au), (av), (aw), (ax), (ay), (az), (ba), (bb) or (be), wherein the N- and/or C-terminal end has been extended by addition of one or more amino acids. In a preferred embodiment, this aspect of the invention is directed to a single-domain antibody having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53.
A further aspect of the invention is directed to use of an sdAb or fusion protein comprising an sdAb having a binding affinity for the antigen microneme protein 2 (MIC2) in an animal feed or animal feed additive wherein the sdAb is selected from the group consisting of wherein the single-domain antibody comprises a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44,
SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NQ:60, SEQ ID NO:61 ,
SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,
SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70 and SEQ ID NO:71.
An aspect of the invention is directed to use of a fusion protein having a binding affinity for the antigen microneme protein 2 (MIC2) in an animal feed or animal feed additive for the prevention or treatment of a parasitic infection, wherein the fusion protein selected from the group consisting of:
a. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:72; b. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:73; c. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:74; d. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:75; e. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:76; f. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:77; g. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:78; h. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:79; i. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NQ:80; and j. polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence
identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:81.
A further aspect of the invention is directed to a method of reducing the likelihood the spread of Eimeria in animals comprising the use of an sdAb, fusion protein, granule, composition, feed additive, or feed of the invention. A further aspect of the invention is directed to an sdAb or fusion protein comprising an sdAb having a binding affinity for the antigen microneme protein 2 as defined by the invention for the preparation of an animal feed additive or animal feed.
One aspect of the invention is directed to use of a composition, an animal feed, or an animal feed additive for treating, preventing or or reducing the spread of Eimeria and Clostridium perfringens infections of a monogastric animal wherein the composition, the animal feed or the animal feed additive comprises the fusion protein or sdAb of the invention. Similarly, the invention is directed to a method of treating, preventing or or reducing the spread of Eimeria and Clostridium perfringens infections, of a monogastric animal comprising administering to the animal a composition, an animal feed or an animal feed additive comprising the fusion protein or sdAb of the invention. Typically, the animal is a bird, more typically poultry.
In one embodiment, the single-domain antibodies of the invention, or fusion proteins comprising multimer components of the single-domain antibodies, as well as compositions comprising the single-domain antibodies or fusion proteins, and methods using the single-domain antibodies or fusion proteins, are capable of preventing or reducing infection levels of Eimeria or Clostridium perfringens infections in a monogastric animal by at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, compared to infection levels without the treatment by the single-domain antibodies or fusion proteins.
A further aspect is directed to method of reducing the levels coccidiocidal or coccidiostatic agents in animal feed or animal feed additives comprising the use of an sdAb or fuion protein of the invention. This may be a method of lowering the amounts of one or more agents selected from ionophores, such as Maduramicin, Salinomycin, Lasalocid, Monensin, Narasin, Semduramicin; Amprolium, Nicarbazin; Sulfonamides such as Sulphadimidine, Sulphaquinoxaline, Sulphadimethoxine, Sulphanitran and Sulphaguanidine: Ethopabate; Quinolones such as Burquinolate, Decoquinate, Clopidol, Robenidine, Halofuginone and Diclazuri in animal feed comprising the use of use of an sdAb or fusion protein of the invention.
Method of Reducing Antimicrobial Resistance (AMR)
The fusion proteins of the invention are a solution to significantly reduce antimicrobial resistance (AMR). Extensive and arguably unnecessary use of antimicrobial agents has led to AMR development in different pathogenic microorganisms. As stated, one aspect of the invention is directed to a method to control, prevent, and treat infections caused by the Eimeria parasite, particularly in the poultry industry. The poultry industry is in serious need of new techniques to control, prevent, and treat infections caused by parasites.
The fusion proteins of the invention can reduce the need for anticoccidial agents in the poultry industry. The fusion proteins of the invention inactivate virulence factors by blocking their binding site, which means the virulence factor is unable to bind. A virulence factor is a protein of a pathogenic bacteria, virus, fungi, or parasite that initiates or facilitates infection. Without virulence factors, the pathogen would not be harmful since it is not able to cause infection. Different types of virulence factors exist: toxins which are secreted virulence proteins, and adhesins which are virulence proteins on the surface of the pathogen.
Without being bound to a particular theory, the mode of action of fusion proteins involves their role as virulence inactivating proteins. The fusion proteins of the invention are proteins which strengthen the gut microbiome and reduce the risk of gastrointestinal infections. The fusion proteins of the invention bind extracellular virulence factors in the gastrointestinal tract so per se they do not directly kill or inhibit growth of pathogens. The fusion protein binding inactivates virulence factors, and as a result, the pathogen is not able to establish a gastrointestinal infection and symptoms are minimized. The fusion protein is designed to bind to and block a particular binding site, active site, or receptor of a specific virulence factor from a pathogen. The desired binding site to be blocked is highly conserved, and if the pathogen mutates, it will lose its binding affinity and is not able to interact with or infect the host anymore. The mutation is thus a loss of function mutation. If the microorganism tries to escape the blocking of its virulence factor by mutating the specific binding site blocked, the virulence factor will lose function as it is not be able to bind to the binding site of the host cell. A benefit from the use of fusion protein of the invention is a reduced use of antimicrobial agents, and the occurrence and spreading of antimicrobial resistance will decrease because the selective pressure on microorganisms is reduced.
The invention fusion proteins of the invention are used to reduce antimicrobial resistance in animals. Without being bound to a particular therory, the virulence factors of pathogens initiate infection in the host. Pathogens have different types of virulence factors: toxins that are excreted, and adhesins that are surface bound. The different types of virulence factors in different pathogens initiate and accelerate infection in the host. A pathogen with inactivated virulence factors can live in a host without infecting the host. Still without being bound to a particular theory, the mode of action of fusion proteins is to block binding sites of virulence factors, thereby inactivating virulence, without interacting intracellularly with the pathogen. The fusion proteins of the invention physically block the binding site of the virulence factor, disabling the virulence factor from binding to the host cell receptor, thereby preventing the pathogen from establishing an infection as intended.
Without being bound to a particular theory, the selective pressure that antimicrobial agents induce, which drives mutations in microorganisms making them resistant, is not the same stress as fusion proteins induce. The difference is that the mode of action of antimicrobial agents is to kill the microorganisms or inhibit the growth of them intracellularly. The microorganisms are attacked and only mutants with resistance surivive. Resistant variants are selected for and will
expand through clonal expansion. The mode of action of fusion proteins is to block the binding site of the virulence factor of the microorganism with high affinity extracellularly. The microorganisms are not attacked, and resistant variants will not be selected for.
A further aspect of the invention is directed to a method of reducing antimicrobial resistance in animals, such as in poultry farming, suitably comprising a method of reducing antimicrobial resistance in an animal population comprising blocking binding sites of virulence factors, thereby disabling the virulence factor from binding to the host cell receptor, thereby preventing the pathogen from establishing an infection, said method comprising administering to said animal a feed or feed additive comprising a single-domain antibody or fusion protein of the invention.
Polynucleotides, nucleic acid constructs, expression vectors and host cells
The present invention also relates to methods of producing a single-domain antibody of the present invention, comprising (a) cultivating a cell, under conditions conducive for production of the single-domain antibody; and optionally, (b) recovering the single-domain antibody.
According to the invention, a method of production of the fusion proteins comprises the use of a polynucleotide encoding a signal peptide which is operably linked to a polynucleotide encoding a fusion protein selected from the group consisting of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80 and SEQ ID NO:81 , which is heterologous to the signal peptide.
According to the invention, a method of production of single-domain antibodies comprises the use of a polynucleotide encoding a signal peptide which is operably linked to a polynucleotide encoding a single-domain antibody selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID
NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166,
SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID
NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210,
SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID
NO:234, SEQ ID NO:238 and SEQ ID NO:242.
An aspect of the invention is directed to a polynucleotide encoding a propeptide operably linked to the polynucleotide of the invention, encoding a single-domain antibody which is heterologous to the propeptide.
The invention is further directed to a nucleic acid construct or expression vector comprising the polynucleotide encoding a fusion protein selected from the group consisting of
SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NQ:80 and SEQ ID N0:81. The nucleic acid construct or expression vector may comprise a gene encoding a protein operably linked to the polynucleotide wherein the gene is heterologous to the polynucleotide encoding the propeptide.
The invention is further directed to a nucleic acid construct or expression vector comprising a polynucleotide encoding a single-domain antibody selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ
ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ
ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ
ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114,
SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242. The nucleic acid construct or expression vector may comprise a gene encoding a protein operably linked to the polynucleotide wherein the gene is heterologous to the polynucleotide encoding the propeptide.
A further aspect is directed to a recombinant host cell comprising a nucleic acid construct or expression vector of the invention. The invention is directed to a method of producing a singledomain antibody or fusion protein of the invention, comprising cultivating the recombinant host cell comprising a nucleic acid construct or expression vector of the invention under conditions conducive for production of the protein. The method typically further comprises recovering the protein.
The invention is furthermore directed to a recombinant host cell comprising a nucleic acid construct or expression vector of the invention and to a method of producing a protein, comprising cultivating the recombinant host cell under conditions conducive for production of the protein. The method typically further comprises recovering the protein. The present invention also relates to methods of producing a single-domain antibody of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the single-domain antibody; and optionally, (b) recovering the single-domain antibody.
The host cell is cultivated in a nutrient medium suitable for production of the single-domain antibody using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state, and/or microcarrier-based fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the single-domain antibody to be expressed
and/or isolated. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the single-domain antibody is secreted into the nutrient medium, the single-domain antibody can be recovered directly from the medium. If the single-domain antibody is not secreted, it can be recovered from cell lysates.
The single-domain antibody may be detected using methods known in the art that are specific for the antibodys, including, but not limited to, the use of specific antigens, DELFIA and ELISA.
The single-domain antibody may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a whole fermentation broth comprising the singledomain antibody is recovered. In another aspect, a cell-free fermentation broth comprising the single-domain antibody is recovered.
The single-domain antibody may be purified by a variety of procedures known in the art to obtain substantially pure single-domain antibodies and/or single-domain antibody fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science-, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10). In an alternative aspect, the single-domain antibody is not recovered.
The polynucleotide may also be mutated by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the single-domain antibody, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107. In an aspect, the polynucleotide is isolated. In another aspect, the polynucleotide is purified.
The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
The polynucleotide may be manipulated in a variety of ways to provide for expression of the single-domain antibody. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
Promoters
The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a single-domain antibody of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the
single-domain antibody. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular single-domain antibodies either homologous or heterologous to the host cell.
Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a bacterial host cell are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY, Davis et al., 2012, supra, and Song et a!., 2016, PLOS One 11(7): e0158447.
Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor and Trichoderma cells, such as the promoters described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
For expression in a yeast host, examples of useful promoters are described by Smolke et al., 2018, “Synthetic Biology: Parts, Devices and Applications” (Chapter 6: Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae), and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
Terminators
The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3’-terminus of the polynucleotide encoding the single-domain antibody. Any terminator that is functional in the host cell may be used in the present invention.
Preferred terminators for bacterial host cells may be obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
Preferred terminators for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
mRNA Stabilizers
The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacterid. 177: 3465-3471).
Examples of mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Ce// 5(11): 1838-1846.
Leader Sequences
The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5’-terminus of the polynucleotide encoding the single-domain antibody. Any leader that is functional in the host cell may be used.
Suitable leaders for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12): 3051-3059, and by Kaberdin and Blasi, 2006, FEMS Microbiol. Rev. 30(6): 967-979.
Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
Suitable leaders for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
Polyadenylation Sequences
The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the polynucleotide which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease. Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.
Signal Peptides
The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a single-domain antibody and directs the single-domain
antibody into the cell’s secretory pathway. The 5’-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the singledomain antibody. Alternatively, the 5’-end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence. A heterologous signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a heterologous signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance secretion of the singledomain antibody. Any signal peptide coding sequence that directs the expressed single-domain antibody into the secretory pathway of a host cell may be used.
Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alphaamylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Freudl, 2018, Microbial Cell Factories 17: 52.
Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptide described by Xu et al., 2018, Biotechnology Letters 40: 949-955
Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.
Propeptides
The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a single-domain antibody. The resultant single-domain antibody is known as a proenzyme or prosingle-domain antibody (or a zymogen in some cases). A prosingle-domain antibody is generally inactive and can be converted to an active singledomain antibody by catalytic or autocatalytic cleavage of the propeptide from the prosingledomain antibody. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a single-domain antibody and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence. Additionally or
alternatively, when both signal peptide and propeptide sequences are present, the single-domain antibody may comprise only a part of the signal peptide sequence and/or only a part of the propeptide sequence. Alternatively, the final or isolated single-domain antibody may comprise a mixture of mature single-domain antibodies and single-domain antibodies which comprise, either partly or in full length, a propeptide sequence and/or a signal peptide sequence.
Regulatory Sequences
It may also be desirable to add regulatory sequences that regulate expression of the single-domain antibody relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In fungal systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals.
Transcription Factors
The control sequence may also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding single-domain antibody that controls the rate of the transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. The transcription factor may function alone and/or together with one or more other single-domain antibodies or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by comprising at least one DNA-binding domain which often attaches to a specific DNA sequence adjacent to the genetic elements which are regulated by the transcription factor. The transcription factor may regulate the expression of a protein of interest either directly, i.e., by activating the transcription of the gene encoding the protein of interest by binding to its promoter, or indirectly, i.e., by activating the transcription of a further transcription factor which regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the further transcription factor. Suitable transcription factors for fungal host cells are described in WO 2017/144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011 , Subcellular Biochemistry 52: 7-23, as well in Balleza et al., 2009, FEMS Microbiol. Rev. 33(1): 133-151.
The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the single-domain antibody at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the single-domain antibody or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ).
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a single-domain antibody. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a single-domain antibody of the present invention.
A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the single-domain antibody and its source. The single-domain antibody can be native or heterologous to the recombinant host cell. Also, at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the single-domain antibody. The recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotide of the present invention.
The host cell may be any microbial cell useful in the recombinant production of a singledomain antibody of the present invention, e.g., a prokaryotic cell or a fungal cell.
The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Grampositive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. In a preferred embodiment, the prokaryotic host cell is E.coli.
The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In an embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus subtilis cell.
For purposes of this invention, Bacillus classes/genera/species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70: 406-438.
The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.
The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.
Methods for introducing DNA into prokaryotic host cells are well-known in the art, and any suitable method can be used including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, with DNA introduced as linearized or as circular polynucleotide. Persons skilled in the art will be readily able to identify a suitable method for introducing DNA into a given prokaryotic cell depending, e.g., on the genus. Methods for introducing DNA into prokaryotic host cells are for example described in Heinze et al., 2018, BMC Microbiology 18:56, Burke et al., 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294, Choi et al., 2006, J. Microbiol. Methods Q4: 391-397, and Donald et al., 2013, J. Bacteriol. 195(11): 2612- 2620.
The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
Fungal cells may be transformed by a process involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation as reviewed by Li et al., 2017, Microbial Cell Factories 16: 168 and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, Christensen etal., 1988, Bio/TechnologyQ: 1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27: 53-75. However, any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as linearized or as circular polynucleotide.
The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, e.g., a Pichia pastoris cell (Komagataella phaffii). In an aspect, the host cell is isolated. In another aspect, the host cell is purified.
The following are preferred embodiments of the invention:
Preferred Embodiments
1. A single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56.
2. The single-domain antibody according to embodiment 1 comprising a CDR3 region having at least 80% sequence identity, such as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96, 97, 98, or 99% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 SEQ ID NO:56.
3. The single-domain antibody according to any of embodiments 1 or 2, wherein the CDR3 region has at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96, 97, 98, or 99% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, and SEQ ID NO:56.
4. The single-domain antibody according to any of embodiments 1 to 3, wherein the CDR3 region has at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, or at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96, 97, 98, or 99% sequence identity, to a sequence selected from the group consisting of SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:48 and SEQ ID NO:56.
5. The single-domain antibody according to embodiment 1 , comprising a CDR3 region a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:56, such as a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, and SEQ ID NO:56, typically a sequence selected from the group consisting of SEQ ID NQ:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:48 and SEQ ID NO:56.
6. The single-domain antibody according to any of embodiments 1 to 5, further comprising a CDR1 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NQ:10, SEQ ID NO:14, SEQ ID NO:18, SEQ
ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:50 and SEQ ID NO:54.
7. The single-domain antibody according to any of embodiments 1 to 6, further comprising a CDR2 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NQ:10, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51 and SEQ ID NO:55.
8. A single-domain antibody having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53.
9. The single-domain antibody of embodiment 8, comprising, consisting essentially of, or consisting of a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, preferably SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, more preferably SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53.
10. A single-domain antibody having a binding affinity for the antigen microneme protein 2 (MIC2), such as a binding affinity for one or more of MIC2 from Eimeria tenella (EtMIC2), MIC2 from Eimeria maxima (EmMIC2), and MIC2 from Eimeria acervulina EaMIC2.
11 . The single-domain antibody according to embodiment 10, having CDR3 as defined in any one embodiments 1 to 5.
12. The single-domain antibody according to embodiment 10 or 11 , having a Kd for EaMIC2 of less than 10 nM.
13. The single-domain antibody according to any of embodiments 10 to 12, having a Kd for EaMIC2 of less than 10 nM and a CDR3 having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:24 and SEQ ID NO: 59 of Family 10 and SEQ ID NO:28, SEQ ID NQ:60 and SEQ ID NO: 61 of Family 13.
14. The single-domain antibody according to embodiment 10 or 11 , having a Kd for EmMIC2 of 10 nM or less, such as of 8 nM or less, such as 7, 6, 5, 4 3, 2, or 1 nM or less.
15. The single-domain antibody according to embodiment 14 having a Kd for EmMIC2 of less than 10 nM wherein the CDR3 region has at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 57 and SEQ ID NO: 58 of Family 8 and SEQ ID NO:20, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO: 64 of Family 21 .
16. The single-domain antibody according to embodiment 10 or 11 , having cross-reactivity by binding to at least two of EaMIC2, EmMIC2 and EtMIC2
17. The single-domain antibody according to embodiment 16 having cross-reactivity by binding to at least two of EaMIC2, EmMIC2 and EtMIC2 and comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, and SEQ ID NO:71 of Family 23.
18. The single-domain antibody according to any of embodiments 10 to 17, further comprising a CDR1 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NQ:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NQ:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NQ:50 and SEQ ID NO:54.
19. The single-domain antibody according to any of embodiments 10 to 18, further comprising a CDR2 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NQ:10, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51 and SEQ ID NO:55.
20. The single-domain antibody according to any of embodiments 10 to 19, having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, preferably SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49 and SEQ ID NO:53, more preferably SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53.
21. A fusion protein comprising two or more sdAbs as defined by any of embodiments 1-20.
22. The fusion protein according to embodiment 21 comprising a homodimer or heterodimer of two sdAb as defined by any of embodiments 1-20.
23. The fusion protein according to any of embodiments 21 to 22 wherein the linker is a polypeptide.
24. The fusion protein according to any of embodiments 21 to 23 wherein the linker is (GGGGS)3.
25. A fusion protein selected from the group consisting of: a. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:72; b. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:73; c. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:74; d. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:75; e. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:76; f. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:77; g. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence
identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:78; h. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:79; i. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NQ:80; and j. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:81.
26. The fusion protein according to any of embodiments 21 to 25 having a binding affinity of 10 nM or less, such as of 8 nM or less, such as 7, 6, 5, 4 3, 2, or 1 nM or less
27. The fusion protein according to any of embodiments 21 to 26 having a retained binding affinity after thermal treatment under method A, of at least 60%, such as at least 65%, such as at least 75%.
28. A composition comprising an sdAb as defined by any of embodiments 1 to 20.
29. A composition comprising the fusion protein as defined by any of embodiments 21 to 27.
30. The composition according to embodiments 28 or 29, further comprising a preservative, filler and/or carrier material.
31. A granule, which comprises:
(a) a core comprising the single-domain antibody of any one of embodiments 1 to 20 or the fusion protein of any of embodiments 21 to 27, and optionally,
(b) a coating consisting of one or more layer(s) surrounding the core.
32. A granule, which comprises:
(a) a core, and
(b) a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises the single-domain antibody of any one of embodiments 1 to 20 or the fusion protein of any of embodiments 21 to 27.
33. A liquid composition comprising the single-domain antibody of any one of embodiments 1 to 20 or the fusion protein of any of embodiments 21 to 27, and a stabilizer, e.g., a polyol such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
34. The liquid composition of embodiment 33, further comprising a filler or carrier material.
35. The liquid composition of paragraph 32 or 33, further comprising a preservative.
36. A composition comprising the single-domain antibody of any one of embodiments 1 to 20 or the fusion protein of any of embodiments 21 to 27, the granule of embodiments 31 or 32, or the liquid composition of any one of embodiments 33 to 35.
37. The composition of embodiment 36, which is a liquid composition, solid composition, solution, dispersion, paste, powder, granule, granulate, coated granulate, tablet, cake, crystal, crystal slurry, gel or pellet.
38. Use of a fusion protein as defined by any of embodiments 21 to 27 or an sdAb as defined by any of embodiments 1 to 20 for the preparation of an animal feed additive.
39. A feed additive comprising an sdAb as defined by any of embodiments 1 to 20 or the fusion protein as defined in any of embodiments 21 to 27.
40. A feed comprising a feed additive as defined by embodiment 39.
41. A method of reducing the likelihood of the spread of Eimeria comprising the use of a granule, composition, feed additive, or feed defined by any of embodiments 28 to 40
42. A method of reducing antimicrobial resistance in an animal population comprising blocking binding sites of virulence factors, thereby disabling the virulence factor from binding to the host cell receptor, thereby preventing the pathogen from establishing an infection, said
method comprising administering to said animal a feed or feed additive comprising a singledomain antibody as defined in any of embodiments 1 to 20 or fusion protein as defined by any of embodiments 21 to 27.
43. A method of increasing the feed conversion ration (FCR) or body weight gain in animals comprising administering to said animal a feed or feed additive comprising a single-domain antibody as defined in any of embodiments 1 to 20 or fusion protein as defined by any of embodiments 21 to 27.
44. A method of treating or preventing coccidiosis in poultry comprising the use of a granule, composition, feed additive, or feed defined by any of embodiments 28 to 40
45. Use of a fusion protein as defined by any of embodiments 21 to 27 or an sdAb as defined by any of embodiments 1 to 20 for the prevention of a medicament for the prevention or treatment of coccidiosis in poultry.
46. A method of reducing parasites or reducing the risk of infections in a pet, such as but not limited to a dog, cat, bird or rabbit.
47. A polynucleotide encoding the single-domain antibody of any one of embodiments 1-20 or fusion protein of any of embodiments 21 to 27.
48. A nucleic acid construct or expression vector comprising the polynucleotide of embodiment 47, operably linked to one or more control sequences that direct the production of the single-domain antibody in an expression host.
49. A recombinant host cell comprising the nucleic acid construct or expression vector of embodiment 48.
50. A method of producing a single-domain antibody having binding activity, comprising cultivating the recombinant host cell of embodiment 49 under conditions conducive for production of the single-domain antibody.
51. A single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NQ:60, SEQ
ID N0:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70 and SEQ ID N0:71.
52. The single-domain antibody according to embodiment 51 , further comprising a CDR1 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NQ:30, SEQ ID NO:42, SEQ ID NQ:50, SEQ ID NO:54, SEQ ID NQ:107, SEQ ID NO:111 , SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:147, SEQ ID NO:159, SEQ ID NO:163, SEQ ID NO:183, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:199, SEQ ID NO:211 , SEQ ID NO:215 and SEQ ID NO:239 SEQ ID NO:2, SEQ ID NO:6, SEQ ID NQ:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NQ:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NQ:50 and SEQ ID NO:54.
53. The single-domain antibody according to embodiment 51 or 52, further comprising a CDR2 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:39, SEQ ID NO:51 , SEQ ID NO:136, SEQ ID NO:164, SEQ ID NO:172, SEQ ID NQ:180, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:192, SEQ ID NO:212 and SEQ ID NO:236 SEQ ID NO:3, SEQ ID NO:7, SEQ ID NQ:10, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51 and SEQ ID NO:55.
54. A single-domain antibody having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, and SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242.
55. A fusion protein comprising two or more sdAbs as defined by any of embodiments 51-54.
56. A fusion protein as defined by any of embodiments 21-27 or 54, or an sdAb as defined by any of embodiments 1-20 or 51-53, for use as a medicament.
57. The fusion protein of embodiment 56, for use in the prevention or treatment of coccidiosis in poultry.
58. A single-domain antibody, fusion protein, composition or method as defined in any of the previous embodiments, wherein administration of the antibody, fusion protein or composition or use of the method is capable of preventing or reducing infection levels of Eimeria or Clostridium perfringens infections in a monogastric animal by at least 25%, such as at least 30%, such as at least 40%, such as at least 50%.
59. An aqueous suitable for adding to drinking water for an animal such as a monogastric animal, wherein the aqueous solution comprises a single-domain antibody or fusion protein as defined in any of the previous embodiments.
60. A method for treating, preventing, or reducing the spread of Eimeria or Clostridium perfringens infections in an animal such as a monogastric animal, the method comprising administering to the animal drinking water comprising a single-domain antibody or fusion protein as defined in any of the previous embodiments.
Examples
Example 1. Screening of single-domain antibodies.
The in vitro binding capacity of various single-domain antibody monomers to recombinant MIC2 protein was tested by a time-resolved fluorescence normalized assay (DELFIA). Briefly, black 96-well immunoblot plates were coated with 2.5 pl/ ml of anti-FLAG M2 antibody (SIGMA #F3165) in phosphate buffered saline (PBS) pH 7.4 overnight (O/N) at 4 °C. After blocking with milk (3% in PBS) for 1 hour at room temperature, the supernatant of single-domain antibody cultures was added at GD600=10 in 6% milk-PBS and incubated at room temperature for 1 hour. After washing, biotinylated antigen (MIC2) was added at 50 nM in 3% milk-PBS and incubated for 1 h at room temperature. After washing, streptavidin-conjugated europium (Perkin Elmer, #1244-360) was added dilute 1/500 in DELFIA assay buffer (Perkin Elmer #1244-111) and incubated for 30 minutes at room temperature. After washing, europium fluorescence was activated using DELFIA enhancement solution (Perkin Elmer #4001-0010). Fluorescence intensity was determined using a microplate reader measuring emission at 615 nm.
To test temperature stability, proteins were kept in a steam box followed by quantification of binding via DELFIA as described above. To test gastric stability, proteins were subjected to 5
ll/ml pepsin at pH 3.0 for 15 min followed by quantification of binding via DELFIA as described above.
Example 2. Recombinant protein production
For antigens and fusion proteins, the genes encoding the desired products were synthesized as linear DNA fragments with appropriate cloning sites in the 3’ and 5’ ends. These were then sub-cloned into the multiple cloning site of a standard expression vector with Lacl induced expression and kanamycin resistance gene selection. For cloning, restriction cloning was applied with Notl and Ncol used as restriction enzymes. The plasmid was subsequently transformed into an E. coli expression strain and streaked to single colonies. A single colony was picked, outgrown in LB with kanamycin (50 pg/ml), and finally stored as single use glycerol stock aliquots. To avoid destructive mutations, gene inserts were sequenced for each expression construct.
For expression, 5 ml LB with kanamycin (50 pg/ml) was inoculated with glycerol stock and incubated overnight at 37 °C and 250 rpm. The following day overnight culture was transferred to autoinduction media and incubated for 2 hrs at 37 °C and 250 rpm followed by another 22 hrs at 25 °C and 250 rpm. The culture was then harvested via centrifugation at 5.000 g for 15 min. The resulting E. coli biomass was stored at -20 °C.
For purification, E. coli biomass containing target protein was thawed and suspended in ice-cold TES buffer (30 mM Tris-HCI, pH 8.0, 1 mM EDTA, 20% Sucrose, 1xPBS) with protein inhibitor cocktail tablet, lysozyme, and benzonase. After 20 min on ice, the suspension was spun down at 15.000 g for 15 minutes. The supernatant was retained and kept on ice. The pellet was then resuspended in ice-cold 5 mM MgSO4 with protein inhibitor cocktail tablet, lysozyme, and benzonase. The suspension was incubated on ice for 20 minutes, before it was spun down at 15.000 g for 15 minutes. The supernatant was pooled with the retained supernatant and spun down at 21.000 g for 60 minutes. The resulting final supernatant was collected.
Nickel affinity columns for immobilized metal affinity chromatography (IMAC) in a gravitybased setup were used for first step of purification. The column was equilibrated with wash buffer (1xPBS with 200 mM NaCI and 20 mM imidazole, pH 8.0) followed by loading the final supernatant. After the supernatant, wash buffer was used to wash the column, before eluting with elution buffer (1xPBS with 200 mM NaCI and 500 mM imidazole, pH 8.0). The protein was then dialyzed overnight into 1xPBS.
For additional purification ion exchange chromatography (I EX) was applied. Depending on theoretical pl of protein, the protein was dialyzed into a 10 mM Tris-HCI 10 mM NaCI with varying pH. The buffer exchanged protein was then loaded onto a 1 ml HiTrap Q or 1 ml HiTrap SP, again depending on pl. The protein was then eluted with gradient increase of NaCI and stored at -20 °C.
Example 3: Characterization of sdAbs: Binding affinity to purified antigen
Binding affinity of purified sdAbs against target MIC2 antigen (Table 1) was evaluated, first via a standard DELFIA assay (Figure 3).
Following affinity guantification with DELFIA, for the best hits the eguilibrium dissociation constant (KD) between sdAbs and antigen was guantified. See the results in Table 1.
KD values were guantified via bio-layer interferometry (BLI). BLI is dependent on the size of the molecules measured. The smaller the molecule, the lower the signal intensity. Additionally, to accurately determine KD, measurements are performed at sdAb concentrations above and below the global KD value. However, also here, the lower the sdAb concentration, the lower the signal. In effect, this creates a lower threshold for KD determination for sdAbs. For sdAbs, this threshold for KD measurement is around 0.5-1 .0 nM.
As could be expected, the sdAbs for which it is was not possible to determine KD were also generally the sdAbs displaying highest TRF signal in Figure 3. This validates the correlation between TRF signal and binding affinity.
Homodimers of bivalent sdAbs benefit from an avidity effect, and as a result, display even stronger binding to their target molecule. Although affinity of sdAbs was already within limits of success criteria, it was decided to proceed with production of bi-valent sdAbs to explore avidity effects.
Example 4: Expression of Fusion Proteins
Fusion protein expression constructs were designed as homodimers from sdAbs as presented above. A (GGGGS)3-linker was used to link the two sdAb seguences.
Fusion proteins were expressed using a standard expression vector, encoding C-terminal 6xHis-tag and FLAG-tag as well as an N-terminal signal peptide for transfer to the periplasm of E. coli BL21 . As with antigens and sdAbs, also fusion proteins were purified using IMAC and I EX.
Fusion proteins were expressed in the periplasm of an E. coli BL21 strain using standard protein expression methods. These were subseguently purified using IMAC followed by I EX. Fusion proteins were expressed and purified to a minimum of 90% purity.
Example 5: Binding affinity of Fusion Proteins to purified antigen
To compare binding affinity between selected sdAbs and corresponding homodimer fusion proteins, a standard DELFIA assay was used (Figure 5) (note that the TRF signal cannot be compared directly to Figure 3 given a lower concentration of antibody (50 nM) in this assay).
It is readily apparent from Figure 5 that all fusion proteins which showed affinity, also displayed significantly higher affinity for their target antigen compared to the corresponding monovalent sdAbs. KD values were guantified using BLI. KD values for the fusion proteins of the invention have been demonstrated to be lower than 1 nM.
In one aspect of the invention, a desired trait of fusion protein is cross-reactivity, meaning the ability of a fusion protein to bind other MIC2 variants than the one it was panned against. To test for cross-reactivity, a standard DELFIA assay was applied.
As shown in Figure 6, purified fusion proteins were tested against all three purified MIC2 antigens at 1000 nM concentration. Interestingly, fusion proteins that bind EtMIC2 strongly also show binding to EmMIC2. Without being bound to a particular theory, since EtMIC2 at the sequence level is the most divergent of the three MIC2 molecules, this indicates that fusion proteins binding to EtMIC2 are interacting with a highly conserved binding site.
The example shows that, as measured by BLI measurements, fusion proteins of the invention have binding affinities with a Kd of less than 1 nM, the success criterion. Of particular interest is the embodiment where the fusion protein is selected from fusion proteins which display cross-binding to both EtMIC2 and EmMIC2 such as SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75.
Example 6: Binding to live parasites
Fecal material was sampled from 10 different chicken houses. Eimeria spp. oocysts were extracted based on established methods and homogenized in a compact bead mill using glass beads to break the oocyst membrane, releasing the individual sporozoites. Homogenized oocysts were then mixed in equal ratios to represent the broadest possible selection of industrially occurring parasite load. For comparison, a commercially available vaccine (Evalon, HIPRA) comprising attenuated Eimeria spp. oocysts was used. In this vaccine oocysts of five Eimeria spp. species are present.
Fusion proteins showed binding to the oocyst material from both fecal matter and vaccine solution. This very strong in vitro validation for fusion proteins to also bind live Eimeria spp. parasites in vivo was surprising.
Example 7: Thermal Stability
Thermal stability is relevant for the product to withstand harsh processing conditions. To assess this quality, a standard ‘steam box’ assay was performed. Fusion proteins were dried with a SpeedVac™ vacuum concentrator and exposed to conditions mimicking formulations of feed products. Fusion proteins that were dried and treated with steam could then be compared to fusion proteins that were only dried.
To accentuate the findings of the steam box assay, the melting temperature of the fusion proteins were measured on a Panta Prometheus system.
Figure 7 shows the retained binding affinity of fusion proteins in percentage after treatment with ‘Steam box’. Table 2 shows the melting temperature and degree of refolding for 10 fusion proteins.
Table 2
= no refolding, '+’ = 0-20% refolding, “++’ = 20-40% refolding, and “+++’ = 40-60% refolding.
Example 8: In vitro screening assay
This assay evaluates the effectiveness of single-domain antibodies of the invention on limiting the invasion of Eimeria spp. sporozoites in a cell culture model. The present assay involves incubating the antibodies at a concentration of 80 pg/mL with freshly prepared Eimeria tenella sporozoites for a period of 8 hours. While the assay described below was performed using these parameters, it can also be performed using different Eimeria species such as E. acervulina or E. maxima, and using other single-domain antibody concentrations or other incubation times.
Assay procedure:
1. Each single-domain antibody is tested for efficacy against cell invasion separately by one or more Eimeria species. In the present example, sporozoites of E. tenella were used, along with three different single-domain antibodies at concentration of 80 pg/mLm, and with a Monensin (a carboxylic polyether ionophore) as a positive control and a no-additive negative control. The antibodies can be diluted in cell culture media or any standard buffer such as PBS with a pH in the range of 5-9. Media or solutions containing proteases should be avoided.
2. MDBK cells (kidney epithelial cells) are seeded at 2.5 x i0e6 per well and are used in the assay once at 90% confluency.
3. Following incubation of single-domain antibodies with freshly excysted Eimeria sporozoites for an appropriate time, in this case 8 hours, the mixtures (0.5 mL) are transferred onto fresh cultures of MDBK cells (kidney epithelial cells) in 48-well plates and incubated for ~24 h (41 °C and 5% CO2). An appropriate number of freshly excysted Eimeria sporozoites used in this step can vary depending e.g. on the species and may, for example, be in the range of about
1 ,000 to 10,000, such as about 2,500-10,000. In the case of E. tenella sporozoites in the present example, 2,500 sporozoites were used.
4. Following each incubation, the plates are washed with PBS and fixed with ethanol for 10 min. The ethanol is then removed, and the plates are washed twice.
5. An anti-E/meria monoclonal antibody (primary) is added to each well and allowed to react for 24 h, after which the plates are washed twice, and a secondary antibody (FITC- labeled) is added and incubated for 6 h followed by a final washing step.
6. The number of infected cells is then differentially counted using a Keyence BZ- X810 system and the percent invasion is calculated for each well.
7. To assess the number of infected cells, the software takes 20 readings/well.
Results
The results for the three tested single-domain antibodies are shown in Table 3 below, where each result is the average for replicates in four wells. The percentages represent the ratio (in %) of the number of sporozoites inside the cells in a treated well (single-domain antibody or monensin) relative to the number of sporozoites inside the cells in an untreated control well.
Table 3: Percent invasion with Eimeria tenella after treatment with single-domain antibodies or positive control
The results of this experiment illustrate that single-domain antibodies of the invention can have efficacy in preventing or reducing infection levels in a cell model which is comparable to the chemical anti-coccidostat monensin.
Example 9: In vivo efficacy study protocol
Studies may be performed to investigate the efficacy of the single-domain antibodies (or fusion proteins) of the invention against Eimeria spp. in broiler chickens under battery cage conditions.
A suitable study may be conducted as a completely randomized design with the desired number of treatments. An uninfected untreated control (UUC) group is included to monitor the severity of the coccidiosis challenge. The UUC and the infected untreated control groups (one infected untreated control group for each Eimeria sp. challenge) may e.g. include 6 replicates of 7 birds/replicate, and groups receiving single-domain antibodies or fusion proteins may e.g.
include 8 replicates of 7 birds/replicate. Allocation of treatments to cages is random and birds are randomly allocated to each cage so the average body weight per cage is similar for all the cages. The chosen number of birds per replicate (e.g. 7) are randomly assigned to each cage per treatment group on the day of set-up and reduced to five birds per cage on the day of challenge to ensure homologous weights per group.
Coccidiosis is induced by experimental infection of chickens, for example Ross 308 male broiler chickens, via oral gavage, with a field strain of pure Eimeria sp., for example E. tenella, E. maxima or E. acervulina, on day 7 of age, while UUC birds are sham inoculated. Treatment efficacy may be assessed by evaluation of different parameters, for example monitoring intestinal lesion scores and oocyst shedding as primary parameters, and body weight gain and feed intake as secondary parameters. Mortality is monitored throughout the trial to adjust the performance parameters.
The number of oocysts per gram feces (OPG) may be counted according to Cringoli et al. (2004), DOI: 10.1016/j.vetpar.2004.05.021 . Lesions may be assessed by euthanizing e.g. three birds per pen and scoring coccidia lesions in the small intestine and ceca.
Trials may be performed for a total of e.g. 16 days, where the trial may be divided up into trial periods, for example day 0-7, day 7-14, day 14-16, day 7-16 and day 0-16 in the case of a 16-day trial. Average daily gain (ADG), average daily feed intake (ADFI) and the ADFI/ADG ratio may be calculated for all trial periods in the 16-day trial. OPG counts and lesion counts may be made on selected days, for example on one or more of days 12, 13, 14, 15 and 16 in the case of a 16-day trial.
Comparison of measured parameters is conducted among i) an infected untreated control (IUC), and ii) one or more groups receiving the single-domain antibodies.
Single-domain antibodies may suitably be added to a European (wheat based) basal diet, formulated based on Ross 2022 recommendations, and provided to birds ad libitum throughout the trial (from 0-16 days of age).
Feed mixing for the basal diet (in pellet form) may be performed by spraying liquid containing the single-domain antibodies on feed pellets after pelleting and cooling. The antibodies may for example be diluted in plain water to obtain a spraying volume of approximately 2-2.5 ml/kg of pellets, with a batch volume of about 200 ml. This may be used for spraying on e.g. 75-100 kg of pellets, for example using a paddle mixer and a laboratory hose pump. A suitable dose of single-domain antibody or fusion protein may e.g. be determined on the basis of in vitro trials, by dose range finding studies, and/or based on the results of initial trials.
Example 10: In vivo efficacy study
An in vivo proof-of-principle study was performed using the protocol described in Example 9. In this study, chickens were infected with E. acervulina, and the number of lesions in an infected control group and in a group treated with the single-domain antibody of SEQ ID NO: 25
was determined on day 14. The single-domain antibody was administered applied on feed pellets which were provided to the birds ad libitum as described in Example 9. The results, expressed as numbers of birds in the two groups with 0, 1 , 2 or 3 lesions per bird, are provided in Table 4. Table 4: Lesion scores in in vivo trial with chickens infected with E. acervulina
It was found in this proof-of-principle study that chickens infected with E. acervulina and treated with the single-domain antibody of SEQ ID NO: 25 had significantly lower lesion score values than chickens in the untreated group.
The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will prevail.
Claims
1. A single-domain antibody comprising a CDR3 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NQ:40, SEQ
ID NO:44, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NQ:60, SEQ
ID NO:61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ
ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70 and SEQ ID NO:71.
2. The single-domain antibody according to claim 1 , further comprising a CDR1 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NQ:30, SEQ ID NO:42, SEQ ID NQ:50, SEQ ID NO:54, SEQ ID NQ:107, SEQ ID NO:111 , SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:147, SEQ ID NO:159, SEQ ID NO:163, SEQ ID NO:183, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:199, SEQ ID NO:211 , SEQ ID NO:215 and SEQ ID NO:239.
3. The single-domain antibody according to any of claims 1 to 2, further comprising a CDR2 region having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:39, SEQ ID NO:51 , SEQ ID NO:136, SEQ ID NO:164, SEQ ID NO:172, SEQ ID NQ:180, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:192, SEQ ID NO:212 and SEQ ID NO:236.
4. A single-domain antibody having at least 75% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NQ:90, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NQ:102, SEQ ID NQ:106, SEQ ID NQ:110, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:126, SEQ ID NQ:130, SEQ ID NO:134, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NQ:150, SEQ ID NO:154, SEQ ID NO:158, SEQ ID NO:162, SEQ ID NO:166, SEQ ID NQ:170, SEQ ID NO:174, SEQ ID NO:178, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NQ:190, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NQ:202, SEQ ID NQ:206, SEQ ID NQ:210, SEQ ID NO:214, SEQ ID NO:218, SEQ ID NO:222, SEQ ID NO:226, SEQ ID NQ:230, SEQ ID NO:234, SEQ ID NO:238 and SEQ ID NO:242.
5. A fusion protein comprising two or more sdAbs as defined by any of claims 1 to 4.
6. A fusion protein selected from the group consisting of: a. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:72; b. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:73; c. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:74; d. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:75; e. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:76; f. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:77; g. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:78; h. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:79; i. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NQ:80; and
j. a polypeptide having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, such as 96%, 97%, 98%, 99% or 100% sequence identity to a polypeptide comprising SEQ ID NO:81.
7. A feed additive comprising an sdAb as defined by any of claims 1 to 4 or the fusion protein as defined in any of claims 5 to 6.
8. A method of reducing the likelihood of the spread of Eimeria comprising the use of a feed additive defined by claim 7.
9. A method of reducing antimicrobial resistance in an animal population comprising administering to said animal a feed or feed additive comprising a single-domain antibody defined by any of claims 1 to 4 or the fusion protein as defined in any of claims 5 to 6.
10. The method of claim 9, wherein antimicrobial resistance is reduced by blocking binding sites of virulence factors, thereby disabling the virulence factors from binding to host cell receptors, thereby preventing pathogens from establishing an infection.
11. A method of increasing the feed conversion ration (FCR) or body weight gain in animals comprising administering to said animal a feed or feed additive comprising a single-domain antibody defined by any of claims 1 to 4 or the fusion protein as defined in any of claims 5 to 6.
12. A method of treating or preventing coccidiosis in poultry comprising the use of a feed additive defined by claim 7.
13. Use of a fusion protein as defined by any of claims 5 to 6 or an sdAb as defined by any of claims 1 to 4 for the preparation of a medicament for the prevention or treatment of coccidiosis in poultry.
14. A fusion protein as defined by claim 5 or 6 or an sdAb as defined by any of claims 1-4 for use as a medicament, e.g. for the prevention or treatment of coccidiosis in poultry.
15. A method of reducing parasites or reducing the risk of infections in a pet, such as but not limited to a dog, cat, bird or rabbit, comprising the use of a feed additive defined by claim 7, or a medicament defined by claim 13.
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