Single Domain Antibodies that Bind and Neutralize Venezuelan Equine Encephalitis Virus

Abstract

Described herein are single-domain antibodies that might serve as alternatives to conventional monoclonal antibodies for either the detection or treatment of Venezuelan equine encephalitis virus (VEEV).

Claims

1. An isolated antibody comprising a protein sequence selected from the group consisting of SEQ ID NOs. 1-8.

2. The isolated antibody of claim 1, wherein the protein sequence comprises exactly two sdAbs joined via a polypeptide linker.

3. A single domain antibody comprising three diverse complementarity determining regions (CDRs) identified as CDR1, CDR2, and CDR3, the antibody having an amino acid sequence with at least 70% identity to a sequence selected from the group consisting of SEQ ID NOs. 1-8 and an amino acid sequence identity in the CDR1, CDR2, and CDR3 regions of at least 75%; and wherein the antibody has binding activity against Venezuelan equine encephalitis virus (VEEV).

4. A single domain antibody of claim 3, further comprising one or more additional single domain antibodies joined thereto together via polypeptide linker(s).

5. A method of treating Venezuelan equine encephalitis virus (VEEV) comprising: obtaining a sample from a patient wherein the sample is known or suspected to contain VEEV; contacting the sample with a single domain antibody under conditions that permit antigen binding thereto; wherein at least a portion of any VEEV in the sample remains bound to the antibody, thereby producing a response indicative of the presence of VEEV in the sample; and if VEEV is detected in the sample, then providing the patient with suitable treatment against VEEV infection, wherein the single domain antibody comprises three diverse complementarity determining regions (CDRs) identified as CDR1, CDR2, and CDR3, the antibody having an amino acid sequence with at least 70% identity to a sequence selected from the group consisting of SEQ ID NOs. 1-8 and an amino acid sequence identity in the CDR1, CDR2, and CDR3 regions of at least 75%.

6. The method of claim 5, wherein the antibody is captured on a surface prior to said contacting.

7. The method of claim 5, wherein the antibody is used as a reporter.

8. The method of claim 5, wherein the single domain antibody comprises exactly two sdAbs joined via a polypeptide linker.

9. The method of claim 5, wherein the patient is a non-human animal.

10. A medicament comprising a single domain antibody of claim 3 and a pharmaceutically-acceptable carrier.

11. A nucleic acid encoding a single domain antibody according to claim 3.

12. The nucleic acid of claim 11, encoding a single domain antibody according to claim 1.

13. The nucleic acid of claim 11, further encoding one or more additional second single domain antibodies according to claim 3, wherein the single domain antibodies are joined together with peptide linkers in a single open reading frame.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] FIG. 1 is a schematic showing antibodies and recombinant derivatives. Other than sdAb, all those depicted exhibit a multi-domain nature. The heavy variable domains are shown with grid shading, light domains are lightly shaded with horizontal lines, and the constant domains is unshaded.

[0019] FIG. 2 provides a sequence alignment of sdAb that bind VEEV. These sdAb fall into five different families based on similarities in their complementarity-determining region three (CDR3) sequences, which occurs at numbered positions 97-114.

[0020] FIGS. 3A and 3B show data from assays in a plaque reduction neutralization test (PRNT) format, shows the ability of sdAbs and linked fusions of certain of the sdAbs, respectively, to inhibit infection of cells by the TC83 strain of VEEV.

DETAILED DESCRIPTION

Definitions

[0021] Before describing the present invention in detail, it is to be understood that the terminology used in the specification is for the purpose of describing particular embodiments, and is not necessarily intended to be limiting. Although many methods, structures and materials similar, modified, or equivalent to those described herein can be used in the practice of the present invention without undue experimentation, the preferred methods, structures and materials are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0022] As used herein, the singular forms “a”, “an,” and “the” do not preclude plural referents, unless the content clearly dictates otherwise.

[0023] As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

[0024] As used herein, the term “about” when used in conjunction with a stated numerical value or range denotes somewhat more or somewhat less than the stated value or range, to within a range of ±10% of that stated.

[0025] A “subject” as used herein is preferably a mammal, for instance, an equine, a rodent, e.g., a mouse, a rat, or a non-human-primate, or a human.

Overview

[0026] Described herein are several single domain antibodies (sdAb; also known as nanobodies or VHH) that bind to Venezuelan equine encephalitis virus (VEEV). These sdAb have the potential to be used in therapeutic applications and can also be used for detection of the virus in immunoassays.

[0027] The general sequences of sdAb are composed of three diverse complementarity determining regions (CDRs) and four more conserved framework regions (FRs). The epitope binding is determined mainly by CDR3.

Examples

[0028] A llama was immunized using an equine vaccine (West Nile Innovator+VEWT) that contained killed Venezuelan equine encephalitis virus (VEEV). Following a series of immunizations, total RNA isolated from the llama's peripheral blood mononuclear cells (PBMCs) was purified. This total RNA was used to create a phage display library of the variable heavy domains of the llama heavy chain only antibodies.

[0029] Five sequence families of anti-VEEV sdAb were selected from the immune phage display library, with a sequence family defined as a group of sdAb sharing a high degree of similarity in the CDR3 region, as seen in FIG. 2. Herein, we define CDR1 as region of amino acid residues numbered 26-35 in FIG. 2, CDR 2 as those numbered 50-65, and CDR3 as those numbered 97-114.

[0030] The VEEV specific binders were isolated by a biopanning procedure on immobilized killed VEEV (Strain TC-83). The selected sequences were subcloned for protein preparation. A subset of prepared sdAb were then subjected to the binding assays and found to be positive for binding TC-83 by Magplex assay.

[0031] The specific amino acid sequences for the group of anti-VEEV sdAb selected from biopanning and the analyses of the representative clones are the subject of this disclosure. Anti-VEEV amino acid sequences are described as follows:

TABLE-US-00001 (V2B3) SEQ ID NO: 1 EVQLQASGGGLVQAGGSLRLSCAASGSIVSINVMAWYRQSPGKERELVAK SSGPFTLYADSVKGRFTISNDAAKNTVDLQMNSLKPEDTAVYYCNAEGLR YPSGTYGPSAVWGQGTQVTVSS (V8C3) SEQ ID NO: 2 EVQLQASGGGLVQAGGSLKVSCAASGRTFSSLAMAWFREAPGNEREFVAA IMWTGDRTHYADFVKGRFTISRDNALNTVSLQMNNLKPADTAVYYCAGAF SFPSQFARDYTYWGQGTQVSVSS (V11A1) SEQ ID NO: 3 DVQLQASGGGSVQAGGSLRLSCVASQNLFEYYTMGWYRQVPGSQRERVAL INNGGSNVAGSVEGRFTISKDNAKNSIYLQMNNLKPEDSAVYYCRAFGPA DYWGQGTQVTVSS (V3G9) SEQ ID NO: 4 EVQLQASGGGLVQAGGSLKVSCAASGRTFNNLAMAWFREAPENEREFVAA IMWTGDRTHYADFVKGRYTISRDNALNTVSLQMNNLKPADTAVYYCAGAF SFPSQFARDYTYWGQGTQVSVSS (V2G5) SEQ ID NO: 5 EVQLQASGGGLVQAGGSLRLSCAASGSIVSINVMAWYRQSPGKQRELVAK SSGPFTLYADSVKGRFTISNDAAKNTVDLQMNSLKPEDTAVYYCNAEGLR YPSGTYGPSAVWGQGTQVTVSS (V2C3) SEQ ID NO: 6 DVQLQASGGGSVQAGGSLRLSCVASQNLFEYYTMGWYRQVPGSQRERVAL INNGGSNVAGSVEGRFTISRDNAKNSIYLQMNNLKPEDSAVYYCRAFGPA DYWGQGTQVTVSS (V2G1) SEQ ID NO: 7 DVQLQASGGGLVQAGDSLRLSCAASGRTIKGYAVGWFRQASGKEREFVAV ISYFDERADYAHSAEGRFTISRDNAKDTVVLQMNSLKPEDTAVYFCAAGL SESTLPSEYIYWGQGTQVSVSS (V3A8) SEQ ID NO: 8 DVQLQASGGGSVQAGGSLRLSCAASGHSFANYIIVAWIRQTPGKECEFVG SASRRDDSTYYADFALGRFIISRDNDINTAYLQMNSLKPEDSAVYYCVAA VMAQTTQGWTTDYDLRGQGTQVTVSS

[0032] Also depicted in FIG. 2 is the consensus sequence having SEQ ID NO: 12 as follows:

TABLE-US-00002 XVQLQASGGGSVQAGGXVQAGXSLXXSCXASXXXXXXXXXXWXRXXXGXX XEXVXXXXXXXXXXXXXAXXXXGRFXISXDXXXXXXXLQMNXLKPXDXAV YXCXXXXXXXXXXXXXXXXXXGQGTQVXVSS

[0033] Several of the sdAb were tested for virus neutralization in a plaque reduction neutralization test (PRNT) where added sdAb is able prevent the infection of cell by the virus. The average of the duplicates was used to calculate 50% and 80% plaque reduction neutralization titer (PRNT50 and PRNT80). Table 1 in FIG. 3A shows the ability of each sdAb to inhibit the TC83 strain of VEEV in PRNT assays.

[0034] Also examined were genetic fusions of the sdAb include where two individual sdAb were joined through a polypeptide linker. For example V2B3 and a derivative of V3A8 termed V3A8f (SEQ ID NO: 9) were linked through a glycine-serine based linker having SEQ ID NO: 13 to produce V2B3-V3A8f (sequence SEQ ID NO: 10) and V3A8f-V2B3 (SEQ ID NO: 11). The sequences of each of these are provided below.

TABLE-US-00003 (V3A8f) SEQ ID NO: 9 DVQLQASGGGSVQAGGSLRLSCAASGHSFANYHVAWIRQTPGKEREFVGS ASRRDDSTYYADFALGRFIISRDNDINTAYLQMNSLKPEDSAVYYCVAAV MAQTTQGWTTDYDLRGQGTQVTVSS (V2B3-V3A8f) SEQ ID NO: 10 EVQLQASGGGLVQAGGSLRLSCAASGSIVSINVMAWYRQSPGKERELVAK SSGPFTLYADSVKGRFTISNDAAKNTVDLQMNSLKPEDTAVYYCNAEGLR YPSGTYGPSAVWGQGTQVTVSSAAAGGGGSGGGGSGGGGSGSDVQLQASG GGSVQAGGSLRLSCAASGHSFANYHVAWIRQTPGKEREFVGSASRRDDST YYADFALGRFIISRDNDINTAYLQMNSLKPEDSAVYYCVAAVMAQTTQGW TTDYDLRGQGTQVTVSS (V3A8-V2B3) SEQ ID NO: 11 DVQLQASGGGSVQAGGSLRLSCAASGHSFANYHVAWIRQTPGKEREFVGS ASRRDDSTYYADFALGRFIISRDNDINTAYLQMNSLKPEDSAVYYCVAAV MAQTTQGWTTDYDLRGQGTQVTVSSAAAGGGGSGGGGSGGGGSGSEVQLQ ASGGGLVQAGGSLRLSCAASGSIVSINVMAWYRQSPGKERELVAKSSGPF TLYADSVKGRFTISNDAAKNTVDLQMNSLKPEDTAVYYCNAEGLRYPSGT YGPSAVWGQGTQVTVSS SEQ ID NO: 13 (linker): AAAGGGGSGGGGSGGGGSGS

[0035] Several other fusions were made similarly. In each case, the fusion involved two sdAb joined by the linker of SEQ ID NO: 13 as in the above examples. Also tested was a sdAb termed CC3 sdAb, which was previously disclosed in commonly-owned U.S. Pat. No. 11,028,152 and has the following sequence (SEQ ID NO: 14):

TABLE-US-00004 EVQLQASGGGSVQAGGSLRLSCVTSQNLFEYYTMGWYRQVPGSQRERVAL INNGGSTVAGSVEGRFTISRDHAKNSVYLQMNYLKPEDSAVYYCRAFGPA DYWGQGTQVTVSS
The CC3 sdAb was earlier identified as binding to chikungunya virus, an alphavirus related to VEEV. Because the anti-VEEV library revealed sequences closely related to CC3, tests included this sdAb as well.

[0036] Table 2 in FIG. 3B provides comparative data on the ability of these linked structures to neutralize VEEV strain TC83 compared to the monomeric sdAb or a cocktail of the two sdAb. Accordingly, aspects of the disclosure include anti-VEEV antibodies that comprise fusions of two or more individual peptide sequences as described above, optionally with a spacer sequence between them.

Further Embodiments

[0037] It is expected that these sdAb and their derivatives could be used in diagnostic assays. For example, a sample known or suspected to contain VEEV could be contacted with a bound or immobilized antibody that includes a protein sequence as described herein under conditions that permit antigen binding thereto. After rinsing the antibody—antigen complex to remove unbound components, wherein at least a portion of any VEEV in the sample remains bound to the antibody, a response indicative of the presence of VEEV in the sample can be obtained by addition of a second anti-VEEV sdAb tagged with biotin that can be recognized by streptavidin-phycoerythrin for completion of a sandwich fluoroimmunoassay. Use of the antibodies in both capture (bound or immobilized) and/or reporter (for example, tagged with a biotin) format is contemplated. Numerous alternative assay formats known in the art could be realized, such as surface plasmon resonance, MagPlex fluorimmunoassay, enzyme-linked immunosorbent assays (ELISA), and the like.

[0038] Variations of the above-described examples are possible. For example, any two individual sdAb, the same or different, could be joined through a polypeptide linker. It would also be possible to join more than two sdAb together to be expressed as a single fusion protein, for example three, four, five, or even more.

[0039] Polypeptide linkers could be shorter or longer than those used in the examples; for instance a linker might be 1 to 50 amino acids in length, inclusive. The length of the linker can be tuned by routine experimentation. It is expected that linkers comprising primarily glycine and serine will function as desired. Thus contemplated are linkers comprising at least 50% glycine and/or serine.

[0040] Furthermore, one or more sdAb can be joined to another protein in order to provide further functionality. For example, the sdAb(s) could be linked to an enzyme or fluorescent protein to aid in detection assays, and/or to a protein domain (e.g., albumin binding domain) that would enable an increased serum half-life that could be important for therapeutic use. Such joining could be accomplished through the expression of a fusion protein (optionally including a linker as noted above) or through post-translational joining

[0041] Also contemplated are variants of the above-described sdAbs based on the consensus sequence depicted in FIG. 2. Variants might be programmatically generated, synthesized, and tested for binding using techniques known in the art.

[0042] For administration to subjects, such as humans, one may employ pharmaceutical compositions comprising one or more anti-VEEV antibodies and a pharmaceutically acceptable carrier or excipient. In the present context, the term “pharmaceutically acceptable” means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to that they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art. They preferably are formulated and administered as a sterile solution, although it may also be possible to utilize lyophilized preparations. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, e.g., pH 5.0 to 7.5. The anti-VEEV antibodies typically are in a solution having a suitable pharmaceutically acceptable buffer, and the composition may also contain a salt. Optionally, stabilizing agent may be present, such as albumin. In certain embodiments, detergent is added. In certain embodiments, the anti-VEEV antibodies may be formulated into an injectable preparation.

[0043] Compositions may be administered to a subject, e.g., a human subject or an equine subject. The total dose of anti-VEEV antibodies in a composition for a single administration can, for instance, be about 0.01 μg to much greater levels, particularly for treatment of adult equines (e.g., about 50 grams). Expected human dosages might be in the rage of about 100 mg to about 5 grams. Determining the recommended dose will be carried out by experimentation and is routine for those skilled in the art.

[0044] Administration of the compositions according to the disclosure can be performed using standard routes of administration. Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transcutaneous, or mucosal administration, e.g., intranasal, oral, and the like. In one embodiment, a composition is administered by intramuscular injection.

Advantages

[0045] As compared to traditional antibodies, sdAb are small and have better penetration to tissues and have a greater probability of crossing the blood brain barrier. This can assist in diagnostic and therapeutic applications for the treatment of VEEV infection, particularly to treat or prevent encephalitis. While on the surface scFv have many similarities to sdAb, they have serious limitations: first, their production is much more difficult; second, they tend to multimerize and aggregate upon storage and are less stable to radiolabeling; and third, they are twice the size of sdAb and would have poorer tissue penetration and be less likely to cross the blood brain barrier.

[0046] These new sequences represent rugged reagents that have potential uses as therapeutics and in detection assays. Whereas conventional antibodies will require cold storage and cannot be easily tailored to work optimally with various detection platforms, sdAb being rugged recombinant binding molecules can be engineered to be even more thermally stable if need be, but they naturally have robust stability being able to refold if denatured, and they can also be expressed with a variety of fusion domains to enhance their utility. Fusion constructs based on sdAb can also be advantageous for tailoring the half-life of therapeutic reagents.