Novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof
阅读说明：本技术 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用 (Novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof ) 是由 张军方 于 2020-06-02 设计创作，主要内容包括：本发明涉及医药生物技术领域,具体公开一种新型冠状病毒(SARS-COV-2)刺突蛋白结合分子及其应用。所述结合分子能特异性结合SARS-COV-2的刺突蛋白且包含至少一个免疫球蛋白单一可变结构域。本发明提供的SARS-COV-2-Spike蛋白结合分子能够特异性的结合SARS-COV-2-Spike蛋白,并有效阻断SARS-COV-2-Spike蛋白与人体细胞ACE2受体的结合,进而阻断SARS-COV-2对细胞的感染过程,抑制SARS-COV-2的传染和扩增。(The invention relates to the field of medical biotechnology, and particularly discloses a novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof. The binding molecule is capable of specifically binding to the spike protein of SARS-COV-2 and comprises at least one immunoglobulin single variable domain. The SARS-COV-2-Spike protein binding molecule provided by the invention can specifically bind to SARS-COV-2-Spike protein, and effectively block the binding of SARS-COV-2-Spike protein and human body cell ACE2 receptor, thereby blocking the infection process of SARS-COV-2 to cells and inhibiting the infection and amplification of SARS-COV-2.)
1. A SARS-COV-2 spike protein binding molecule, characterized by: is capable of specifically binding to the spike protein of SARS-COV-2 and comprises at least one immunoglobulin single variable domain, the CDR1, CDR2 and CDR3 of which are selected from any one of the following combinations:
1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3;
2) CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 6;
3) CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8 and CDR3 shown in SEQ ID NO. 9;
4) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11 and CDR3 shown in SEQ ID NO. 12;
5) CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14 and CDR3 shown in SEQ ID NO. 15;
6) CDR1 shown in SEQ ID NO. 16, CDR2 shown in SEQ ID NO. 17 and CDR3 shown in SEQ ID NO. 18;
7) CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20 and CDR3 shown in SEQ ID NO. 21;
8) CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 23 and CDR3 shown in SEQ ID NO. 24;
9) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26 and CDR3 shown in SEQ ID NO. 27;
10) CDR1 shown in SEQ ID NO. 28, CDR2 shown in SEQ ID NO. 29 and CDR3 shown in SEQ ID NO. 30;
11) CDR1 shown in SEQ ID NO. 31, CDR2 shown in SEQ ID NO. 32 and CDR3 shown in SEQ ID NO. 33;
12) CDR1 shown in SEQ ID NO. 34, CDR2 shown in SEQ ID NO. 35 and CDR3 shown in SEQ ID NO. 36;
13) CDR1 shown in SEQ ID NO. 37, CDR2 shown in SEQ ID NO. 38 and CDR3 shown in SEQ ID NO. 39;
14) CDR1 shown in SEQ ID NO. 40, CDR2 shown in SEQ ID NO. 41 and CDR3 shown in SEQ ID NO. 42;
15) CDR1 shown in SEQ ID NO. 43, CDR2 shown in SEQ ID NO. 44 and CDR3 shown in SEQ ID NO. 45;
16) CDR1 shown in SEQ ID NO. 46, CDR2 shown in SEQ ID NO. 47 and CDR3 shown in SEQ ID NO. 48;
17) CDR1 shown in SEQ ID NO. 49, CDR2 shown in SEQ ID NO. 50 and CDR3 shown in SEQ ID NO. 51;
18) CDR1 shown in SEQ ID NO. 52, CDR2 shown in SEQ ID NO. 53 and CDR3 shown in SEQ ID NO. 54;
19) CDR1 shown in SEQ ID NO. 55, CDR2 shown in SEQ ID NO. 56 and CDR3 shown in SEQ ID NO. 57;
20) CDR1 shown in SEQ ID NO. 58, CDR2 shown in SEQ ID NO. 59 and CDR3 shown in SEQ ID NO. 60;
21) CDR1 shown in SEQ ID NO. 61, CDR2 shown in SEQ ID NO. 62 and CDR3 shown in SEQ ID NO. 63;
22) CDR1 shown in SEQ ID NO. 64, CDR2 shown in SEQ ID NO. 65 and CDR3 shown in SEQ ID NO. 66;
23) CDR1 shown in SEQ ID NO. 67, CDR2 shown in SEQ ID NO. 68 and CDR3 shown in SEQ ID NO. 69;
24) CDR1 shown in SEQ ID NO. 70, CDR2 shown in SEQ ID NO. 71 and CDR3 shown in SEQ ID NO. 72;
25) CDR1 shown in SEQ ID NO. 73, CDR2 shown in SEQ ID NO. 74 and CDR3 shown in SEQ ID NO. 75;
26) CDR1 shown in SEQ ID NO. 76, CDR2 shown in SEQ ID NO. 77 and CDR3 shown in SEQ ID NO. 78;
27) CDR1 shown in SEQ ID NO:79, CDR2 shown in SEQ ID NO:80 and CDR3 shown in SEQ ID NO: 81.
2. The SARS-COV-2 spike protein binding molecule of claim 1, wherein: the immunoglobulin single variable domain is a single domain antibody.
3. The SARS-COV-2 spike protein binding molecule of claim 2, wherein: the single domain antibody comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 82-108.
4. The SARS-COV-2 spike protein binding molecule of claim 2, wherein: the single domain antibody comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 82-108.
5. The SARS-COV-2 spike protein binding molecule of claim 2, wherein: the single domain antibody comprises an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NOs: 82-108.
6. The SARS-COV-2 spike protein binding molecule of claim 2, wherein: the single domain antibody comprises any one of the amino acid sequences of SEQ ID NO 82-108.
7. The SARS-COV-2 spike protein binding molecule of any one of claims 1 to 6, wherein: further comprising an immunoglobulin Fc region.
8. The SARS-COV-2 spike protein binding molecule of claim 7, wherein: the immunoglobulin Fc region is a human immunoglobulin Fc region.
9. The SARS-COV-2 spike protein binding molecule of claim 8, wherein: the immunoglobulin Fc region is the Fc region of human IgG 1.
10. The SARS-COV-2 spike protein binding molecule of claim 9, wherein: the amino acid sequence of the immunoglobulin Fc region is SEQIDNO 109.
11. The SARS-COV-2 spike protein binding molecule of claim 10, wherein: comprises at least one amino acid sequence in SEQ ID NO. 110-136.
12. The SARS-COV-2 spike protein binding molecule of any one of claims 1 to 6, 8 to 11, wherein: has at least one of the following features:
a. has KD value less than 1 × 10 for binding with SARS-COV-2 spike protein-8M；
b. Blocking the combination of SARS-COV-2 and human cell receptor ACE 2;
c. inhibit the infection and amplification of SARS-COV-2.
13. The SARS-COV-2 spike protein binding molecule of claim 7, wherein: has at least one of the following features:
a. has KD value less than 1 × 10 for binding with SARS-COV-2 spike protein-8M；
b. Blocking the combination of SARS-COV-2 spike protein and human cell receptor ACE 2;
c. inhibit SARS-COV-2 infection and amplification.
14. A nucleic acid molecule encoding the SARS-COV-2 spike protein binding molecule of any one of claims 1 to 13.
15. An expression vector comprising the nucleic acid molecule of claim 14 and expression control elements thereof.
16. A host cell comprising and expressing the nucleic acid molecule of claim 14.
17. A method of obtaining a SARS-COV-2 spike protein binding molecule according to any one of claims 1 to 13, comprising:
a. culturing the host cell of claim 16 under conditions that allow expression of the SARS-COV-2 spike protein binding molecule;
b. collecting the SARS-COV-2 spike protein binding molecule expressed by the host cell from the culture of step a.
18. An immunoconjugate comprising the SARS-COV-2 spike protein binding molecule of any one of claims 1 to 13 conjugated to a therapeutic moiety.
19. A pharmaceutical composition comprising a SARS-COV-2 spike protein binding molecule of any one of claims 1 to 13 and/or an immunoconjugate of claim 18, and a pharmaceutically acceptable carrier.
20. Use of the pharmaceutical composition of claim 19 in the manufacture of a medicament for the treatment or prevention of pneumonia as a novel coronavirus disease.
21. A kit for detecting SARS-COV-2, comprising a SARS-COV-2 spike protein binding molecule of any one of claims 1 to 13.
22. The method of using the kit of claim 21, wherein the formation of the complex is detected by contacting the test sample and the control sample with the SARS-COV-2 spike protein binding molecule of any one of claims 1 to 13 under conditions such that the complex is formed between the SARS-COV-2 spike protein binding molecule and the SARS-COV-2 spike protein binding molecule of any one of claims 1 to 13; the presence of SARS-COV-2 in the sample is determined by the difference in complex formation between the test sample and the control sample.
Technical Field
The invention relates to the field of medical biotechnology, in particular to a novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof.
Background
The total infection of the novel coronavirus pneumonia (COVID-19) exceeds more than 400 million people in the world, the number of infected people is still rapidly increased, and specific and effective treatment means for the COVID-19 is lacking clinically at present. Although the epidemic situation in China has been comprehensively controlled, the epidemic situation in foreign countries is outbreak and is also rapidly growing. In addition, more and more studies have shown that infection with the novel coronavirus (SARS-COV-2) may present a chronic carrier state; partial discharge of the patient with regaining yang also suggests that the virus may be present in the human body for a long time. At present, the key factors of long-term carrying, such as mechanism, time and the like are not clear, and the prevention of SARS-COV-2 soil rolling is important in the future. Under the influence of COVID-19, economic loss, social burden and other negative effects caused by the COVID-19 are difficult to measure in China and countries all over the world.
At present, no specific medicine exists in COVID-19, and rapid development of effective medicines is urgently needed. Many research and development organizations both at home and abroad have a second conflict in the research of the treatment strategy aiming at the COVID-19. Although the developed broad-spectrum small-molecule antiviral drugs such as Reidcisvir, Favipiravir and the like have certain curative effect on COVID-19, the drug has no specificity to SARS-COV-2 and has limited curative effect, so the drug is difficult to become a specific drug of COVID-19.
Disclosure of Invention
Aiming at the problems that the prior antiviral drug has no specificity to the novel coronavirus of the novel coronavirus, has limited treatment effect and is difficult to become a specific drug for SARS-COV-2, the invention provides a novel coronavirus (SARS-COV-2) spike protein binding molecule and application thereof.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a novel coronavirus (SARS-COV-2) spike protein binding molecule capable of specifically binding to SARS-COV-2 spike protein and comprising at least one immunoglobulin single variable domain, wherein CDR1, CDR2 and CDR3 in said immunoglobulin single variable domain are selected from any one of the following combinations:
1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3;
2) CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 6;
3) CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8 and CDR3 shown in SEQ ID NO. 9;
4) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11 and CDR3 shown in SEQ ID NO. 12;
5) CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14 and CDR3 shown in SEQ ID NO. 15;
6) CDR1 shown in SEQ ID NO. 16, CDR2 shown in SEQ ID NO. 17 and CDR3 shown in SEQ ID NO. 18;
7) CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20 and CDR3 shown in SEQ ID NO. 21;
8) CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 23 and CDR3 shown in SEQ ID NO. 24;
9) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26 and CDR3 shown in SEQ ID NO. 27;
10) CDR1 shown in SEQ ID NO. 28, CDR2 shown in SEQ ID NO. 29 and CDR3 shown in SEQ ID NO. 30;
11) CDR1 shown in SEQ ID NO. 31, CDR2 shown in SEQ ID NO. 32 and CDR3 shown in SEQ ID NO. 33;
12) CDR1 shown in SEQ ID NO. 34, CDR2 shown in SEQ ID NO. 35 and CDR3 shown in SEQ ID NO. 36;
13) CDR1 shown in SEQ ID NO. 37, CDR2 shown in SEQ ID NO. 38 and CDR3 shown in SEQ ID NO. 39;
14) CDR1 shown in SEQ ID NO. 40, CDR2 shown in SEQ ID NO. 41 and CDR3 shown in SEQ ID NO. 42;
15) CDR1 shown in SEQ ID NO. 43, CDR2 shown in SEQ ID NO. 44 and CDR3 shown in SEQ ID NO. 45;
16) CDR1 shown in SEQ ID NO. 46, CDR2 shown in SEQ ID NO. 47 and CDR3 shown in SEQ ID NO. 48;
17) CDR1 shown in SEQ ID NO. 49, CDR2 shown in SEQ ID NO. 50 and CDR3 shown in SEQ ID NO. 51;
18) CDR1 shown in SEQ ID NO. 52, CDR2 shown in SEQ ID NO. 53 and CDR3 shown in SEQ ID NO. 54;
19) CDR1 shown in SEQ ID NO. 55, CDR2 shown in SEQ ID NO. 56 and CDR3 shown in SEQ ID NO. 57;
20) CDR1 shown in SEQ ID NO. 58, CDR2 shown in SEQ ID NO. 59 and CDR3 shown in SEQ ID NO. 60;
21) CDR1 shown in SEQ ID NO. 61, CDR2 shown in SEQ ID NO. 62 and CDR3 shown in SEQ ID NO. 63;
22) CDR1 shown in SEQ ID NO. 64, CDR2 shown in SEQ ID NO. 65 and CDR3 shown in SEQ ID NO. 66;
23) CDR1 shown in SEQ ID NO. 67, CDR2 shown in SEQ ID NO. 68 and CDR3 shown in SEQ ID NO. 69;
24) CDR1 shown in SEQ ID NO. 70, CDR2 shown in SEQ ID NO. 71 and CDR3 shown in SEQ ID NO. 72;
25) CDR1 shown in SEQ ID NO. 73, CDR2 shown in SEQ ID NO. 74 and CDR3 shown in SEQ ID NO. 75;
26) CDR1 shown in SEQ ID NO. 76, CDR2 shown in SEQ ID NO. 77 and CDR3 shown in SEQ ID NO. 78;
27) CDR1 shown in SEQ ID NO:79, CDR2 shown in SEQ ID NO:80 and CDR3 shown in SEQ ID NO: 81.
Compared with the prior art, the SARS-COV-2 Spike protein (SARS-COV-2-Spike protein) binding molecule provided by the invention can specifically bind to SARS-COV-2-Spike protein, effectively block the binding of SARS-COV-2-Spike protein and human body cell ACE2 receptor, further block the infection process of SARS-COV-2 to cells, and inhibit the infection and amplification of SARS-COV-2. The SARS-COV-2-Spike protein binding molecule provided by the invention also has the characteristics of good specificity of binding with SARS-COV-2-Spike protein, high biological activity and stability and no toxic or side effect.
Preferably, the immunoglobulin single variable domain is a single domain antibody.
Preferably, the single domain antibody comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 82-108.
Preferably, the single domain antibody comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 82-108.
Preferably, the single domain antibody comprises an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NOs 82-108.
The amino acid sequence of the single domain antibody comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared with any one of SEQ ID NO: 82-108.
Preferably, the single domain antibody comprises any one of the amino acid sequences of SEQ ID NO 82-108.
Preferably, the SARS-COV-2 spike protein binding molecule further comprises an immunoglobulin Fc region.
The inclusion of an immunoglobulin Fc region in the SARS-COV-2 spike protein binding molecules of the invention allows the binding molecules to form dimers while further extending the in vivo half-life of the molecule. The Fc region useful in the present invention may be from different subtypes of immunoglobulin, for example, IgG (IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM.
Preferably, the immunoglobulin Fc region is a human immunoglobulin Fc region.
Preferably, the immunoglobulin Fc region is the Fc region of
Preferably, the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO. 109.
Preferably, it comprises at least one amino acid sequence of SEQ ID NO: 110-136.
The stability and biological activity of the combined molecule fused with the Fc region are further improved, and the KD value of the combined molecule combined with SARS-COV-2 spike protein is further reduced.
Preferably, the SARS-COV-2 spike protein binding molecule has at least one of the following characteristics:
a. has KD value less than 1 × 10 for binding with SARS-COV-2 spike protein-8M；
b. Blocking the combination of SARS-COV-2 and human
c. inhibit the infection and amplification of SARS-COV-2.
The invention also provides a nucleic acid molecule for coding the SARS-COV-2 spike protein binding molecule, wherein the nucleic acid molecule is RNA, DNA or cDNA, which can be obtained by artificial synthesis or separated from proper natural sources.
The invention also provides an expression vector containing the nucleic acid molecule and an expression control element thereof. The expression vector typically comprises at least one nucleic acid molecule provided herein operably linked to one or more suitable expression regulatory elements (promoters, enhancers, terminators, integration factors, selection markers, leaders, reporters, and the like). The selection of such elements and their sequences for expression in a particular host cell is within the knowledge of one skilled in the art.
The invention also provides host cells comprising and expressing the nucleic acid molecules. The host cell is a cell for expressing a heterologous protein, including a bacterial cell, a fungal cell, or a mammalian cell.
The invention also provides a method for obtaining the SARS-COV-2 spike protein binding molecule, which comprises the following steps:
a. culturing the above host cell under conditions that allow expression of the SARS-COV-2 spike protein binding molecule;
b. collecting the SARS-COV-2 spike protein binding molecule expressed by the host cell from the culture of step a.
The recombination of specific nucleic acid molecules into expression vectors and expression into host cells by transformation or transfection methods, selection of markers, methods of inducing protein expression, culture conditions, and the like are known in the art. Techniques for the isolation and purification of protein binding molecules are well known to those skilled in the art.
The SARS-COV-2 spike protein binding molecules of the invention can also be obtained by other methods known in the art for producing proteins, such as chemical synthesis.
The invention also provides an immunoconjugate comprising a SARS-COV-2 spike protein binding molecule of any one of the above conjugated to a therapeutic moiety.
The invention also provides a pharmaceutical composition comprising the SARS-COV-2 spike protein binding molecule and/or the immunoconjugate described above, and a pharmaceutically acceptable carrier.
The "pharmaceutically acceptable carrier" according to the present invention includes any solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic and absorption delaying agent, and the like which are physiologically compatible. The carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the binding molecule, immunoconjugate, may be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound, as is well known to those skilled in the art.
The pharmaceutical composition of the invention may further comprise other adjuvants and auxiliary materials, etc. according to the needs.
The invention also provides application of the pharmaceutical composition in preparing a medicine for treating or preventing novel pneumonia caused by the coronavirus disease.
The invention also provides a kit for detecting SARS-COV-2, comprising any one of the SARS-COV-2 spike protein binding molecules.
The invention also provides the using method of the kit for detecting SARS-COV-2, under the condition that the SARS-COV-2 spike protein binding molecule and the SARS-COV-2 spike protein can form a compound, the SARS-COV-2 spike protein binding molecule is contacted with a detection sample and a control sample, and the formation of the compound is detected; the presence of SARS-COV-2 in the sample is determined by the difference in complex formation between the test sample and the control sample.
Drawings
FIG. 1 is an agarose gel electrophoresis of total RNA extracted in example 1 of the present invention, wherein M: DNA marker2000, lane 1: total RNA;
FIG. 2 is an agarose gel electrophoresis of the Step1-PCR amplification product of nested PCR-amplified single domain antibody gene in example 1 of the present invention, wherein M: DNA marker2000, lane 1: (ii) amplification products;
FIG. 3 is an agarose gel electrophoresis of Step2-PCR amplification product of nested PCR amplification of single domain antibody gene in example 1 of the present invention, wherein DNA marker2000,
FIG. 4 is an agarose gel electrophoresis image of the SfiI and Not1 double cleavage products of the target single domain antibody gene and the vector pHEN1 in example 1 of the present invention, wherein, DNA marker2000, lane 1: pHEN 1; lane 2: pHEN1 after the enzyme digestion of sfil/notI; lane 3: a single domain antibody gene after sfil/notI enzyme digestion;
FIG. 5 is an agarose gel electrophoresis of colony PCR amplification products for determining library insertion rates in example 1 of the present invention, wherein M: DNA marker 2000; lanes 1-48: 48 colonies were picked;
FIG. 6 is a graph showing the change in viral load of rhesus monkeys in the treatment group and the control group according to the change in days in example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The following examples are provided to better illustrate the embodiments of the present invention.
Definition of
Unless otherwise indicated or defined, all terms used have the ordinary meaning in the art that will be understood by those skilled in the art. Moreover, unless otherwise indicated, all methods, steps, techniques and operations not specifically recited may be and have been performed in a manner known per se to those of skill in the art.
Unless otherwise indicated, the terms "antibody" or "immunoglobulin" used interchangeably herein, whether referring to a heavy chain antibody or a conventional 4-chain antibody, are used as a general term to include full-length antibodies, individual chains thereof, as well as all portions, domains, or fragments thereof (including but not limited to antigen-binding domains or fragments). Furthermore, as used herein, the term "sequence" (e.g., in the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "single domain antibody sequence", or "protein sequence", etc.) should generally be understood to include both the relevant amino acid sequences and the nucleic acid or nucleotide sequences encoding the sequences, unless a more limited interpretation is required herein.
The term "immunoglobulin variable domain" as used herein refers to an immunoglobulin domain consisting essentially of four "framework regions" referred to in the art and hereinafter as "
Conventional IgG antibody molecules generally consist of a light chain comprising 1 variable region (VL) and 1 constant region (CL) and a heavy chain comprising 1 variable region (VH) and 3 constant regions (CH1, CH2, CH 3). Single domain antibodies (sdabs), which are antibodies lacking the light chain of the antibody and having only the variable region of the heavy chain, are also called nanobodies (nanobodies) because of their small molecular weight. Single domain antibodies specifically bind epitopes without the need for additional antigen binding domains. Single domain antibodies are small, stable and efficient antigen recognition units formed from a single immunoglobulin domain.
The total number of amino acid residues in each CDR may be different as is known in the art for single domain antibodies.
The total number of amino acid residues in a single domain antibody will generally range from 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.
Other structural and functional properties of single domain antibodies and polypeptides containing the same can be summarized as follows:
single domain antibodies (which have been naturally "designed" to functionally bind to an antigen in the absence and without interaction with a light chain variable domain) can be used as a single and relatively small functional antigen binding unit, domain or polypeptide. This distinguishes single domain antibodies from the VH and VL domains of conventional 4 chain antibodies, which are themselves generally unsuitable for practical use as single antigen binding proteins or immunoglobulin single variable domains, but need to be combined in some form or another to provide a functional antigen binding unit (e.g. in the form of a conventional antibody fragment such as a Fab fragment; or in the form of an scFv consisting of a VH domain covalently linked to a VL domain).
Because of these unique properties described above, the use of single domain antibodies or as part of larger polypeptides offers many significant advantages over the use of conventional VH and VL domains, scfvs or conventional antibody fragments (e.g., Fab-or F (ab') 2-fragments), e.g., single domain antibodies require only a single domain to bind antigen with high affinity and high selectivity, such that neither two separate domains need be present, nor are there a need to ensure that the two domains are present in the proper spatial conformation and configuration (e.g., scfvs typically require the use of specially designed linkers); single domain antibodies can be expressed from a single gene and do not require post-translational folding or modification; single domain antibodies can be easily engineered into multivalent and multispecific formats; single domain antibodies are highly soluble and have no tendency to aggregate; single domain antibodies are highly stable to heat, pH, proteases and other denaturants or conditions, and therefore can be prepared, stored or transported without the use of refrigeration equipment, thereby achieving cost, time and environmental savings; single domain antibodies are easy to prepare and relatively inexpensive, even on the scale required for production; single domain antibodies are relatively small compared to conventional 4 chain antibodies and antigen binding fragments thereof (about 15kDa or 1/10 of conventional IgG in size), and therefore exhibit higher tissue permeability and can be administered at higher doses compared to conventional 4 chain antibodies and antigen binding fragments thereof; single domain antibodies may exhibit so-called cavity binding properties (especially due to their extended CDR3 loops compared to conventional VH domains) allowing access to targets and epitopes not accessible by conventional 4 chain antibodies and antigen binding fragments thereof.
Methods for obtaining single domain antibodies that bind to a particular antigen or epitope have been previously disclosed in the following references: r. van der Lindenet, journal of immunologicalcalels methods,240(2000) 185-); liatal, jbiolchem, 287(2012) 13713-13721; deffatet, African journal of Biotechnology Vol.8(12), pp.2645-2652,17June,2009 and WO 94/04678.
In addition, those skilled in the art will also appreciate that it is possible to "graft" one or more of the above CDRs onto other "scaffolds," including but not limited to human scaffolds or non-immunoglobulin scaffolds. Scaffolds and techniques suitable for such CDR grafting are known in the art.
In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen binding molecule or antigen binding protein (e.g., an immunoglobulin single variable domain of the invention) molecule can bind. Specificity of an antigen-binding molecule can be determined based on its affinity and/or avidity. The affinity, expressed by the dissociation equilibrium constant (KD) of an antigen to an antigen binding protein, is a measure of the strength of binding between an epitope and the antigen binding site on the antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen-binding molecule (alternatively, affinity can also be expressed as the association constant (KA), which is 1/KD). As will be appreciated by those skilled in the art, affinity can be determined in a known manner depending on the particular antigen of interest. Avidity is a measure of the strength of binding between an antigen binding molecule (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and an associated antigen. Affinity is related to both: affinity to its antigen binding site on the antigen binding molecule, and the number of relevant binding sites present on the antigen binding molecule.
The term "SARS-COV-2 Spike protein binding molecule (SARS-COV-2-Spike protein binding molecule)" as used herein means any molecule capable of specifically binding to SARS-COV-2 Spike protein. The SARS-COV-2 spike protein binding molecule may comprise a single domain antibody or conjugate thereof as defined herein directed against the SARS-COV-2 spike protein. SARS-COV-2 spike protein binding molecules also encompass so-called "SMIP" ("small modular immunopharmaceuticals"), or immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules.
The "SARS-COV-2 spike protein binding molecule" of the invention may comprise at least one immunoglobulin single variable domain, such as a single domain antibody, that binds to the SARS-COV-2 spike protein. In some embodiments, a "SARS-COV-2 spike protein binding molecule" of the invention can comprise two immunoglobulin single variable domains, such as single domain antibodies, that bind to the SARS-COV-2 spike protein. SARS-COV-2 spike protein binding molecules containing more than one immunoglobulin single variable domain are also known as "formatted" SARS-COV-2 spike protein binding molecules. The formatted SARS-COV-2 spike protein binding molecule may also comprise, in addition to the immunoglobulin single variable domain that binds to the SARS-COV-2 spike protein, a linker and/or a moiety with effector function, for example a half-life extending moiety (such as an immunoglobulin single variable domain that binds to serum albumin), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as PEG) and/or an Fc region. The "SARS-COV-2 spike protein binding molecule" of the invention also encompasses bispecific antibodies that contain immunoglobulin single variable domains that bind different antigens.
Generally, the SARS-COV-2 spike protein binding molecules of the invention will be preferably 10 as measured in a Biacore or Kin ExA assay-8To 10-12Mole/liter (M), more preferably 10-9To 10-11Mole/liter, even more preferably 10-10To 10-12Even more preferably 10-11To 10-12Or a lower dissociation constant (KD). Any greater than 10-4The KD value of M is generally considered to indicate non-specific binding. Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, Surface Plasmon Resonance (SPR) assays, as described herein, and/or competitive binding assays (e.g., Enzyme Immunoassay (EIA) and sandwich competitive assays).
Amino acid residues will be represented according to the standard three-letter or one-letter amino acid code as is well known and agreed upon in the art. Such conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are preferably made where one amino acid within the following groups (1) - (5) is replaced with another amino acid residue within the same group: (1) smaller aliphatic nonpolar or weakly polar residues: ala, Ser, Thr, Pro, and Gly; (2) polar negatively charged residues and their (uncharged) amides: asp, Asn, Glu and Gln; (3) polar positively charged residues: his, Arg and Lys; (4) larger aliphatic non-polar residues: met, Leu, Ile, Val and Cys; and (5) aromatic residues: phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: ala substituted by Gly or Ser; arg is replaced by Lys; asn is replaced by Gln or His; asp substituted by Glu; cys is substituted with Ser; gln is substituted by Asn; glu is substituted with Asp; gly by Ala or Pro; his is substituted with Asn or Gln; ile is substituted by Leu or Val; leu is substituted by Ile or Val; lys is substituted with Arg, Gln, or Glu; met is substituted by Leu, Tyr or Ile; phe is substituted by Met, Leu or Tyr; ser substituted by Thr; thr is substituted by Ser; trp is substituted by Tyr; tyr is substituted with Trp or Phe; val is substituted by Ile or Leu.
"sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity. For the determination of sequence identity see, for example: sequence analysis molecular biology, von heinje, g., academic press,1987 and sequence analysis primer, Gribskov, m.and devereux, j., eds., mstockton press, new york, 1991.
A polypeptide or nucleic acid molecule is considered "substantially isolated" when it has been separated from at least one other component with which it is normally associated in the source or medium (culture medium), such as another protein/polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or minor component, as compared to the reaction medium or culture medium from which it is naturally derived and/or from which it is obtained. In particular, a polypeptide or nucleic acid molecule is considered "substantially isolated" when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold and up to 1000-fold or more than 1000-fold. The "substantially isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous, as determined by suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).
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