ANTI-FIBROBLAST ACTIVATION PROTEIN (FAP) SINGLE DOMAIN ANTIBODIES AND USES THEREOF

Technical field

The invention relates to the field of biomedical technology, and more particularly to antifibroblast activation protein (FAP) single domain antibodies (sdAbs) and uses thereof.

Background technique

Fibroblast activation protein alpha (FAP), also known as Separse, is a 170 kDa single pass type II transmembrane glycoprotein belonging to the dipeptidyl peptidase 4 family. It is highly expressed in cancer-associated fibroblasts (CAFs) and plays an important roles in modulating the tumour microenvironment and supports the tumours cells through the release of enzymes, cytokines and growth factor.

Studies have shown that FAP expression is highly upregulated on reactive CAFs of more than 90% of all primary and metastatic epithelial tumors, while it is generally absent from normal adult tissues. FAP overexpression is associated with poor prognosis and increased risk of metastasis. The expression level of FAP in human has also been evaluated in 28 different kinds of cancers with ⁶⁸ Ga-FAPI PET/CT. The ⁶⁸ Ga-FAPI uptake varies across and within different tumor entities while the background in muscle and blood pool is low. The highest SUV _max (>12) is found in sarcoma, esophageal, breast, cholangiocarcinoma, and lung cancer while intermediate SUV _m ax (6-12) is found in hepatocellular, colorectal, head-neck, ovarian, pancreatic, and prostate cancer. Epidemiologically important tumor entities demonstrates selective high update present an unique opportunity to target FAP for cancer diagnostic and therapeutic (Kratochwil et al., JNM, 2019, 60:801-805).

Multiple FAP targeted monoclonal antibodies, antibody conjugates and combination drugs have been raised for development for clinical purposes. Sibrotuzumab/BIBHl, a humanized version of the F19 antibody that specifically binds to human FAP (described in WO 99/57151) were development. A Phase I study result showed that ¹³¹ 1 Sibrotuzumab was well tolerated and safe. While Sibrotuzumab received FDA approval for phase II, the early phase II trial of unconjugated sibrotuzumab in patients with metastatic colorectal cancer was discontinued due to the lack of efficacy of the antibody in inhibiting tumor progression (Hofheinz et al., Onkologie 26, 44-48 (2003)).

Single domain antibody (sdAb) or nanobody is camel variable domain of heavy chain of heavy-chain antibody (VHH). It is the smallest antibody binding domain with molecular weight of 1 / 10 of that of a conventional antibody. In addition to the antigenic reactivity of monoclonal antibodies, sdAbs also have some unique functional characteristics, such as low molecular weight, strong stability, good solubility, easy expression, weak immunogenicity, strong penetration, strong targeting, simple humanization, low production cost etc. Unique structure of sdAbs also makes them suitable for carrying radioisotope. Single domain antibodies can penetrate the tumor tissue binding target quickly and specifically, while the non-binding sdAbs can be quickly removed from the blood and reduce the radiation dose of the body. Compared to conventional antibodies, sdAbs have more advantages as tracers and targeted internal radiotherapy drugs.

Therefore, there is an urgent need to develop FAP targeted sdAb which could bind to FAP with high specificity and efficiency for the development of next generation of radioimmunoimaging and radioimmunotherapy.

Summary of the Invention

The object of the present invention is to provide a class of specific single domain antibodies (sdAbs) that are effective against FAP.

In the first aspect of the present invention, it provides an anti-FAP single domain antibody, which comprises the following three CDRs

CDR1 as shown in SEQ ID No: 5n+3, CDR2 as shown in SEQ ID No: 5n+4, CDR1 as shown in SEQ ID No: 5n+5, wherein n=0, 1, 2, 3, 4, 5, 6, or 7.

In another preferred embodiment, the FAP is human FAP.

In another preferred embodiment, the anti-FAP single domain antibody comprises 4 frame regions (FRs) and 3 complementary determining regions (CDRs), wherein the CDRs comprise the corresponding CDR1, CDR2 and CDR3 as defined above, as well as FR1, FR2, FR3 and FR4 separated by said CDR1, CDR2 and CDR3.

Furthermore, a heavy chain variable region of an anti-human FAP antibody is provided, the heavy chain variable region comprises three complementary determining regions CDR1, CDR2, and CDR3, and the three CDRs comprise the corresponding CDR1, CDR2 and CDR3 as defined hereinabove.

In another preferred embodiment, the three CDRs comprise CDR1, CDR2 and CDR3 as shown in Table IB.

In another preferred embodiment, the anti-FAP single domain antibody is selected from the group consisting of :

(Z 1 ) CDR1 as shown in SEQ ID No: 3, CDR2 as shown in SEQ ID No: 4, CDR3 as shown in SEQ ID No: 5;

(Z2) CDR1 as shown in SEQ ID No: 8, CDR2 as shown in SEQ ID No: 9, CDR3 as shown in SEQ ID No: 10;

(Z3) CDR1 as shown in SEQ ID No: 13, CDR2 as shown in SEQ ID No: 14, CDR3 as shown in SEQ ID No: 15;

(Z4) CDR1 as shown in SEQ ID No: 18, CDR2 as shown in SEQ ID No: 19, CDR3 as shown in SEQ ID No: 20;

(Z5) CDR1 as shown in SEQ ID No: 23, CDR2 as shown in SEQ ID No: 24, CDR3 as shown in SEQ ID No: 25;

(Z6) CDR1 as shown in SEQ ID No: 28, CDR2 as shown in SEQ ID No: 29, CDR3 as shown in SEQ ID No: 30;

(Z7) CDR1 as shown in SEQ ID No: 33, CDR2 as shown in SEQ ID No: 34, CDR3 as shown in SEQ ID No: 35; and

(Z8) CDR1 as shown in SEQ ID No: 38, CDR2 as shown in SEQ ID No: 39, CDR3 as shown in SEQ ID No: 40.

In another preferred embodiment, the anti-FAP single domain antibody is selected from the group consisting of :

(Zl) CDR1 as shown in SEQ ID No: 3, CDR2 as shown in SEQ ID No: 4, CDR3 as shown in SEQ ID No: 5;

(Z2) CDR1 as shown in SEQ ID No: 8, CDR2 as shown in SEQ ID No: 9, CDR3 as shown in SEQ ID No: 10;

In another preferred embodiment, the anti-FAP single domain antibody comprises an amino acid sequence of as shown in any one of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, and 36.

In another preferred embodiment, the anti-FAP single domain antibody has a high affinity for FAP.

In another preferred embodiment, the anti-FAP single domain antibody does not blocking FAP activity.

In the second aspect of the present invention, it provides a polynucleotide an anti-FAP single domain antibody in the first aspect, or a fusion protein containing the anti-FAP single domain antibody.

In another preferred embodiment, the polynucleotide comprises DNA or RNA.

In another preferred embodiment, the polynucleotide has a nucleotide sequence as shown in any one of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, and 37.

In the third aspect of present invention provides an expression vector, the expression vector comprises the polynucleotide according to the second aspect.

In the fourth aspect of the present invention provides a host cell, the host cell comprises the expression vector according to the third aspect, or the polynucleotide according to the second aspect is integrated within the genome of the host cell.

In another preferred embodiment, the host cell includes a prokaryocyte or an eukaryocyte.

In another preferred embodiment, the host cell is selected from the group consisting of E.coli. and yeast cell.

In the fifth aspect of the present invention provides a method for producing an anti-FAP single domain antibody comprising the steps of:

(a) culturing said host cell according to the fourth aspect under a condition suitable for producing a single domain antibody, thereby obtaining a culture containing the anti-FAP single domain antibody; and

(b) isolating or recovering said anti-FAP single domain antibody from said culture.

In the sixth aspect of the present invention, it provides an immunoconjugate, which comprises:

(a) the anti-FAP single domain antibody in the first aspect, or a fusion protein containing the anti-FAP single domain antibody; and (b) a conjugating moiety selected from the group consisting of a detectable marker, drug, toxin, cytokine, radionuclide, and enzyme.

In another preferred embodiment, the conjugating part is a drug or toxin.

In another preferred embodiment, the conjugating part is a detectable marker.

In another preferred embodiment, the conjugate is selected from the group consisting of fluorescent or luminescent markers, radiomarkers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (eg, IL- 2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL), chemotherapeutic agents (eg, cisplatin) or any form of nanoparticles, etc. that can produce detectable products.

In another preferred embodiment, the immunoconjugate contains multivalent (such as bivalent) VHH chains of the anti-FAP single domain antibody according to the first aspect of the present invention. The multivalent refers that the amino acid sequence of the immunoconjugate contains several repeated VHH chains of the anti-FAP single domain antibody according to the first aspect of the present invention.

In the seventh aspect of the invention, it provides a use of the anti-FAP single domain antibody according to the present invention for preparing (a) a reagent for detecting FAP molecule; and/or (b) a medicament for treating cancer.

In another preferred embodiment, the detecting comprises detection conducted by flow cytometry or cell immunofluorescence.

In the eighth aspect of the present invention, it provides a pharmaceutical composition comprising:

(i) the anti-FAP single domain antibody according to the first aspect of the present invention, or a fusion protein containing the anti-FAP single domain antibody, or the immunoconjugate according to seventh aspect of the present invention; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is in a form of injection.

In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating cancers, and the cancer is selected from the group consisting of gastric cancer, liver cancer, leukemia, renal tumor, lung cancer, small intestinal cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor and bladder tumor.

In the ninth aspect of the present invention, it provides one or more uses of the anti-FAP single domain antibody according to the present invention:

(i) for detecting human FAP molecule;

(ii) for flow cytometry assay;

(iii) for cell immunofluorescence detection;

(iv) for treating cancer; (v) for diagnosing cancer.

In another preferred embodiment, the use is non-diagnostic and non-therapeutic.

In the tenth aspect of the present invention, it provides an antibody comprising: the heavy chain variable region VHH according to the first aspect of the present invention.

In another preferred embodiment, the antibody is an antibody specific for the FAP protein.

In another preferred embodiment, the antibody is a nanobody.

In another preferred embodiment, the antibody is a bi-specific or multi-specific antibody.

In the eleventh aspect of the present invention, it provides a recombinant protein, and the recombinant protein has:

(i) a sequence of anti-FAP single domain antibody according to the first aspect of the present invention; and

(ii) an optional tag sequence assisting expression and/or purification.

In another preferred embodiment, the tag sequence includes 6His tag or HA tag.

In another preferred embodiment, the recombinant protein specifically binds to the FAP protein.

In the twelfth aspect of the present invention, it provides a use of the anti-FAP single domain antibody according to the first aspect of the present invention, or a fusion protein thereof, or an immunoconjugate thereof for preparing a medicament, agent, detecting plate or kit; wherein, the agent, detecting plate or kit is used for detecting FAP protein in the sample; wherein, the medicament is used for treating or preventing cancers expressing FAP (i.e. FAP positive).

In another preferred embodiment, the cancer comprises gastric cancer, lymphoma, liver cancer, leukemia, renal tumor, lung cancer, small intestinal cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, or adrenal tumors.

In the thirteenth aspect of the present invention, it provides a method for detecting FAP protein in a sample, which comprises the steps of:

(1) contacting the sample with the anti-FAP single domain antibody according to the second aspect of the present invention, or a fusion protein thereof, or an immunoconjugate thereof;

(2) detecting the antigen-antibody complex, wherein the detected complex indicated the presence of FAP protein.

In the fourteenth aspect of the present invention, it provides a method for treating a disease, comprising administering the anti-FAP single domain antibody, or a fusion protein thereof, or an immunoconjugate thereof to a subject in need.

In another preferred embodiment, the subject comprises mammals, such as human.

It should be understood that in the present invention, the technical features specifically described above and below (such as the Examples) can be combined with each other, thereby constituting a new or preferred technical solution, which will not be redundantly repeated one by one.

Drawings description

Figure 1 shows SDS-PAGE analysis of human FAP -His antigen. The purity of FAP -His was greater than 90%.

Figure 2 shows construction and quality control of the phage display library for anti-FAP single domain antibody. The VHH repertoire amplification was determined by the two-step nested PCR (Figure A). It demonstrated that the obtained VHH gene fragment was approximately 400bp. The transformants of the library was determined by plating the clones in serial dilution (Figure B). It is confirmed that the transformants of the constructed library is 5.2x10 ⁹ cfu. The insertion rate of the library was determined by colony PCR (Figure C). It is verified that the insertion rate of the constructed library is 91.7%. The results of the bio-panning as determined by counting the plate. It is revealed that the constructed library is enriched 44 folds after three rounds of bio-panning.

Figure 3 shows the FACS based binding analysis of the anti-FAP single domain antibodies to FAP expressing glioblastoma cancer cell line U87-MG. It is confirmed that the lead anti-FAP single domain antibodies bind to FAP on the surface of U87-MG.

Detailed Description

After extensive and intensive studies, the inventors have successfully obtained a class of anti-FAP single domain antibodies after numerous screening. The experimental results show that the single domain antibodies are not only high specificity, and can efficiently bind to the FAP molecules on cell lines expressing FAP molecules. It is possible to deliver functional molecules (toxins or small RNAs) by modifying this type of antibody to kill FAP positive cells or perform other functional studies. Based on this discovery, the invention is completed.

In particular, the human FAP protein as antigen was used to immunize a camel, thereby obtaining a gene library of single domain antibodies with high quality. The FAP protein molecules were conjugated onto an ESLIA board and exhibited correct spatial structure of FAP protein. The antigens in such configuration were used to screen the gene library of single domain antibodies by phage exhibition technology (phage exhibition of a gene library of camel heavy chain antibody) thereby obtaining genes of single domain antibodies with FAP specificity. Then the genes were transferred into E. coli thereby obtaining the stains which can be effectively expressed in E.coli with high specificity.

As used herein, the terms "nanobodies of the invention", " anti-FAP single domain antibody of the invention", and "the anti-FAP nanobodies of the invention" are exchangeable and refer to nanobodies that specifically recognize and bind to FAP (including human FAP).

As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycosaminoglycan protein of about 150,000 Dalton with the same structural features, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain through a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isoforms is different. Each heavy and light chain also has intra-chain disulfide bonds which are regular spaced. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light and heavy chains.

As used herein, the terms "single domain antibody (sdAbs)" and "nanobodies" have the same meaning referring to a variable region of a heavy chain of an antibody, and construct a single domain antibody (VHH) consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Generally, the antibodies with a natural deficiency of the light chain and the heavy chain constant region 1 (CHI) are first obtained, the variable regions of the heavy chain of the antibody are therefore cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

As used herein, the term "variable" refers that certain portions of the variable region in the nanobodies vary in sequences, which forms the binding and specificity of various specific antibodies to their particular antigen. However, variability is not uniformly distributed throughout the nanobody variable region. It is concentrated in three segments called complementary-determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chain. The more conserved part of the variable region is called the framework region (FR). The variable regions of the natural heavy and light chains each contain four FR regions, which are substantially in a P-folded configuration, joined by three CDRs which form a linking loop, and in some cases can form a partially P-folded structure. The CDRs in each chain are closely adjacent to the others by the FR regions and form an antigen-binding site of the nanobody with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91 -3242, Volume I, pages 647-669. (1991)). The constant regions are not directly involved in the binding of the nanobody to the antigen, but they exhibit different effects or functions, for example, involve in antibody-dependent cytotoxicity of the antibodies.

As known by those skilled in the art, immunoconjugates and fusion expression products include: conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the nanobodies or fragments thereof of the present invention. The invention also includes a cell surface marker or an antigen that binds to said anti- FAP protein nanobody or the fragment thereof.

As used herein, the term "heavy chain variable region" and "VH" can be used interchangeably.

As used herein, the terms "variable region" and "complementary determining region (CDR)" can be used interchangeably.

In another preferred embodiment, the heavy chain variable region of said nanobody comprises 3 complementary determining regions: CDR1, CDR2, and CDR3.

In another preferred embodiment, the heavy chain of said nanobody comprises the above said heavy chain variable region and a heavy chain constant region.

According to the present invention, the terms "nanobody of the invention", "protein of the invention", and "polypeptide of the invention" are used interchangeably and all refer to a polypeptide, such as a protein or polypeptide having a heavy chain variable region, that specifically binds to FAP protein. They may or may not contain a starting methionine.

The invention also provides other proteins or fusion expression products having the nanobodies of the invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e. immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region are identical or at least 90% identical, preferably at least 95% identical to the heavy chain of the nanobody of the present invention.

In general, the antigen-binding properties of a nanobody can be described by three specific regions located in the variable region of the heavy chain, referred as variable regions (CDRs), and the segment is divided into four frame regions (FRs). The amino acid sequences of four FRs are relatively conservative and do not directly participate in binding reactions. These CDRs form a loop structure in which the 0-sheets formed by the FRs therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the nanobody. The amino acid sequences of the same type of nanobodies can be compared to determine which amino acids constitute the FR or CDR regions.

The variable regions of the heavy chains of the nanobodies of the invention become a particular interest because at least a part of them is involved in binding antigens. Thus, the present invention includes those molecules having a nanobody heavy chain variable region with a CDR, provided that their CDRs are 90% or more (preferably 95% or more, the most preferably 98% or more) identical to the CDRs identified herein.

The present invention includes not only intact nanobodies but also fragment(s) of immunologically active nanobody or fusion protein(s) formed from nanobodies with other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the nanobodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the same biological function or activity of a nanobody of the invention. Polypeptide fragments, derivatives or analogs of the invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably non-conservative amino acid residues) substituted. Such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having a substituent group in one or more amino acid residues; or (iii) a polypeptide formed by fusing a mature polypeptide and another compound (such as a compound that increases the half-life of the polypeptide, for example, polyethylene glycol); or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader or secretory sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6 His tag). According to the teachings herein, these fragments, derivatives, and analogs are within the scope of one of ordinary skill in the art.

The nanobody of the present invention refers to a polypeptide including the above CDR regions having FAP protein binding activity. The term also encompasses variant forms of polypeptides comprising the above CDR regions that have the same function as the nanobodies of the invention. These variations include, but are not limited to, deletion insertions and/or substitutions of one or several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1- 10) amino acids, and addition of one or several (generally less than 20, preferably less than 10, and more preferably less than 5) amino acids at C-terminus and/or N-terminus. For example, in the art, the substitution of amino acids with analogical or similar properties usually does not alter the function of the protein. For another example, addition of one or several amino acids at the C- terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the nanobodies of the invention.

The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNAs capable of hybridizing with DNA encoding the nanobody of the present invention under high or low stringent conditions, and polypeptides or proteins obtained using antiserum against the nanobodies of the invention.

The invention also provides other polypeptides, such as a fusion protein comprising nanobodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of the nanobodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the nanobody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

In the present invention, "a conservative variant of a nanobody of the present invention" refers to the polypeptides in which there are up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids substituted by amino acids having analogical or similar properties, compared to the amino acid sequence of the nanobody of the present invention. These conservative variant polypeptides are preferably produced according to the amino acid substitutions in Table A.

Table A

The present invention also provides a polynucleotide molecule encoding the above nanobody or fragment or fusion protein thereof. Polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

Polynucleotides encoding the mature polypeptides of the invention include: coding sequences only encoding mature polypeptide; coding sequences for the mature polypeptide and various additional coding sequences; coding sequences (and optional additional coding sequences) and non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide that encodes the polypeptide, and may also include a polynucleotide that includes additional coding and/or non-coding sequences.

The invention also relates to polynucleotides that hybridize to the sequences described above and that have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention specifically relates to polynucleotides that can be hybridized to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) additional denaturants during hybridization, such as 50% (v/v) formamide, 0.1% fetal bovine serum I 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only under the identity between the two sequences at least over 90%, preferably over 95%. Also, polypeptides encoded by hybridizable polynucleotides have the same biological functions and activities as mature polypeptides.

The full-length nucleotide sequence of the nanobody of the present invention or a fragment thereof can generally be obtained by a PCR amplification method, a recombination method, or an artificial synthesis method. One possible method is to synthesize related sequences using synthetic methods, especially when the fragment length is short. In general, a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (eg, 6His) can be fused together to form a fusion protein.

Once the concerned sequences have been obtained, the concerned sequences can be obtained in large scale using recombinant methods. Usually, sequences can be obtained by cloning it into a vector, transferring it into cells, and then isolating the sequences from the proliferated host cells by conventional methods. Bio-molecules (nucleic acids, proteins, etc.) to which the present invention relates include bio-molecules that exist in isolated form.

At present, DNA sequences encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) can be obtained completely by chemical synthesis. The DNA sequence then can be introduced into various existing DNA molecules (or e.g. vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising the above-mentioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform an appropriate host cell so that it can express the protein.

The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces, bacterial cells such as Salmonella typhimurium, fungal cells such as yeast, insect cells of Drosophila S2 or SI9, animal cells of CHO, COS7, 293 cells, and the like.

The transformation of the host cell with the recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCh method. The procedures used are well known in the art. Another method is to use MgCh. If necessary, conversion can also be performed by electroporation. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.

The obtained transformants can be cultured in a conventional manner to express the polypeptide encoded by the gene of the present invention. Depending on the host cells used, the medium used in the culture may be selected from various conventional media. The culture is performed under conditions suitable for the host cells growth. After the host cells are grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction) and the cells are incubated for a further period of time.

The recombinant polypeptide in the above method may be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods by utilizing its physical, chemical and other characteristics. These methods are well-known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic disruption, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and the combinations thereof.

The nanobodies of the invention may be used alone or in combination or conjugated with a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or a combination thereof.

Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

Therapeutic agents that can be bound or conjugated to the nanobodies of the present invention include, but are not limited to: 1. Radionuclides; 2. Biological poisons; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposome; 7. Nano magnetic particles; 8. Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

Pharmaceutical composition

The invention also provides a composition. Preferably, said composition is a pharmaceutical composition comprising the above nanobody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier. In general, these materials can be formulated in non-toxic, inert, and pharmaceutically acceptable aqueous carrier media wherein the pH is generally about 5-8, preferably about 6-8, although the pH can be varied with the nature of the formulation material and the condition to be treated. The formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the present invention can be directly used to bind FAP protein molecules and thus can be used to treat tumors. In addition, other therapeutic agents can also be used at the same time.

The pharmaceutical composition of the present invention contains a safe and effective amount (for example, 0.001 -99 wt%, preferably 0.01 -90 wt%, and more preferably 0.1-80 wt%) of the above-mentioned nanobodies of the present invention (or their conjugates) and pharmaceutically acceptable carriers or excipients. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and the combinations thereof. The drug formulation should be suitable for the mode of administration. The pharmaceutical composition of the present invention may be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvant. Pharmaceutical compositions such as injections and solutions are preferably made under aseptic conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 10 micrograms/kilogram body weight to about 50 milligrams/kilogram body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the immune- conjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kilogram body weight, and in most cases, no more than about 50 mg/kilogram body weight, preferably the dose is about 10 micrograms/kilogram body weight to about 10 milligrams/kilogram body weight. Of course, factors such as the route of administration and the patient's health status should be considered to define the specific doses, all of which are within the skills of skilled physicians.

Nanobodies with markers

In a preferred embodiment of the invention, the nanobodies carry detectable markers. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, colored markers, and fluorescent markers.

Colloidal gold markers can be performed using methods known to those skilled in the art. In a preferred embodiment of the invention, the anti-FAP nanobodies are marked with colloidal gold to obtain colloidal gold nanobodies.

The anti-FAP nanobodies of the present invention have very good specificity and high potency.

Detection method

The invention also relates to a method of detecting FAP protein. The method steps are basically as follows: obtaining a sample of cells and/or tissue; dissolving the sample in a medium; and detecting the level of FAP protein in the dissolved sample.

According to the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells which is present in a cell preservation solution.

Kits

The present invention also provides a kit containing a nanobody (or a fragment thereof) or a detection board of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, an instruction, a buffer, and the like.

The present invention also provides a detection kit for detecting the level of FAP, and said kit comprises nanobodies that recognize FAP protein, a lysis medium for dissolving a sample, a general reagent and a buffer needed for the detection, such as various buffer, detection markers, detection substrates, etc. The test kit can be an in vitro diagnostic device.

Application

As described above, the nanobodies of the present invention have extensive biological application value and clinical application value. Said applications involve various fields such as diagnosis and treatment of diseases related to FAP, basic medical research, and biological research. One preferred application is for clinical diagnosis and targeted treatment of FAP.

The main advantages of the present invention include:

(a) the nanobodies of the invention is highly specific to the human FAP protein with correct spatial structure;

(b) the nanobodies of the invention have a strong affinity; and

(c) the nanobodies of the invention are simple to produce.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in which the specific conditions are not indicated in the following examples are usually carried out according to the conditions described in the conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) manufacturing conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight and parts by weight. Unless otherwise stated, the biological materials are commercially available or prepared according to the conventional methods.

Example 1: Expression and purification of human FAP protein

In the example, human FAP extracellular domain protein was transiently expressed in mammalian cell HE293F. Briefly, the nucleotide sequence of human FAP extracellular domain was subcloned into a commerially available pTT5 vector. The recombinant vector was mixed with transfection reagent PEI at a ratio of 1:3 and transformed into HEK293F cells.

The transfected cells was incubated at 37°C, 6% CO2 shaking bed incubator for 6 days. The supernatant of cells was collected and the single domain antibody was purified using Ni-NTA affinity chromatography.

The eluted single domain antibody was ultrafiltrated with PBS and sampled for SDS-PAGE analysis after yield quantification. The purity of FAP-His was greater than 90% (Figure 1) and was subsequently used for camel immunization.

Example 2: Construction and screening of anti-FAP sdAb library

The Camelus bactrianus was immunized against the purified FAP-His antigen once a week for a total of 7 times. The total RNA was extracted from the peripheral blood lymphocytes of the camel at the end of immunization. The cDNA was synthesized and the VHH encoding sequences were amplified by a two-steps nested PCR (Figure 2A). The VHH encoding sequences were subcloned into pMECs phagemid and the recombinant vector was transformed into TGI cells for the construction of phage display library. The transformants of the library was determined by plating the clones in serial dilutions and the insertion rate was determined by randomly selecting 24 clones for colony PCR detection. The results demonstrated that the transformants and insertion rate of the constructed library 5.2xl0 ⁹ cfu(Figure 2B) and 91.7% (Figure 2C) respectively.

The constructed library was enriched about 44 folds after three rounds of bio-panning (Fig. 2D). 600 clones from the constructed library were randomly selected for ELISA with human FAP -his. Positive hits (ratio > 3) were sequenced and 70 distinct single domain antibodies were identified from sequencing.

Example 3: Expression and purification of anti-FAP sdAb

The corresponding plasmids from selected clones (33 clones) in Example 2 were transformed into E.coli WK6 and incubated on LA+glucose medium culture plate overnight at 37 °C. Single colony was picked and inoculated in 5 ml of LB medium containing ampicillin and incubated at 37 °C in shaking bed incubator overnight. 1ml overnight culture was transferred to 330ml TB culture medium and incubated at 37 °C in shaking bed incubator until OD reaching 0.1-1. IPTG was added and the culture was incubated at 37 °C in shaking bed incubator overnight. The culture was collected with centrifugation and the periplasmic extract was purified by osmotic shock. The single domain antibody was further purified using Ni-NTA affinity chromatography.

The purity of single domain antibody was greater than 90% and was subsequently used for functional studies. The following 8 lead single domain antibodies were obtained.

Table 1A Sequence information for anti-FAP sdAbs

The sequence of the 8 lead single domain antibodies are as follow, where the three CDR regions are underlined.

Amino acid sequences:

SEQ ID NO: l(MY8094-5-39)

OVOLOESGGGLVOPGGSLRLSCAASGFGFGNSLMSWVRRAPGKGLEWVSTIYPRG

SFTDYADSVKGRFTISRDNTRSTLYLQMNSLRTEDTAVYYCATGWGRSSDYDPPGOG T QVTVSS

SEQ ID NO: 6(MY8094-2-80)

OVOLOESGGGSVOAGGSLRLSCTASGYTSITYSMAWFROAPGKEREGVAFIRSGGS ITYYADSVKGRFTISRDGPKNTLYLQMNSLKPEDTAMYYCAAGLRGYDGDWYDGTHF RYWGQGTQVTVSS

SEQ ID NO: 11 (MY8094-2-32)

OVOLOESGGGLAOPGGSLRLSCAASGFGFSYSAMNWVROAPGKGLEWVSTIDSRG GATSYADSVKGRFTISRDNAKNTLYLQMNSLKTEDTAMYYCSTGYRAYDNSPRGQGT QVTVSS

SEQ ID NO: 16(MY8094-2-73)

OVOLOESGGGSVOAGGSLRLSCAASGYTYSGNYIVGWFROAPGKEREGVAAIYPY AGGYSTYYGNFVKGRFTISRDTAKNTVYLOMNSLKPEDTAMYYCAADKRGMGSILAV EGYNYWGOGTQVTVS S

SEQ ID NO: 21(MY8094-4-39)

QVOLQESGGGSVOAGGSLRLSCVGSGYMFSTNAMSWVROAPGKELEWVTTITAG GHTGYADSVKGRFTISRDNAKNTLYLOLNSLKTEDTAMYYCANRFYSSDYVARRDFGY WGQGTQVTVSS

SEQ ID NO: 26(MY8094-4-42)

OVOLOESGGGSVOAGGSLKLSCAASGFVFSTNAMSWVROAPGKELEWVTTITAGG HTGYADSVKGRFTISRDNAKNTLYLOLNSLKTEDTAMYYCANRFYSSDYVARRDFGY WGQGTQVTVSS

SEQ ID NO: 31(MY8094-5-5)

OVOLOESGGGSVOAGGSLRLSCAASEYKTTFDTMAWFROAPGKEREGVASIRTRS GLTYYAGSVKGRFTISPDNAKNTVSLQMNDLKPEDTAMYYCATGFRGFLLDTGWSTSA LYDYWGOGTOVTVS S

SEQ ID NO: 36(MY8094-5-74)

OVOLOESGGGLVOPGGSLRLSCAASGFVFSTNAMSWVROAPGKELEWVTTITAGG HTGYADSVKGRFTISRDNAKNTLYLOLNSLKTEDTAMYYCANRFYSSDYVARRNFGY WGQGTQVTVSS

Nucleotide sequences:

SEQ ID NO: 2(MY8094-5-39)

GGAAGTCTATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCG CGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTG CAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGTTTCGGATTCGGGAAC AGCCTCATGAGCTGGGTCCGCCGGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCAAC TATTTACCCGCGTGGAAGTTTCACGGACTATGCGGACTCCGTGAAGGGCCGATTCAC CATCTCCAGAGACAACACCCGGAGCACGCTCTATCTGCAAATGAACAGCCTGAGGA CTGAGGACACTGCCGTGTATTATTGTGCCACAGGTTGGGGACGTTCGAGTGACTATG ATCCCCCGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCATACCCGTAC GACGTTCCGGACTACGGTTCCCACCACCATCACCATCACTAGACTGTTGAAAGTTGT TTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAAC TTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTCGT TTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTAT CCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTG AGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTT ATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATC CTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATAG GTTCCGAAATAGGCAGGGTGCATTAACTGTTTATACGGGCACTGTTACTCAAGGCAC TGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGC

SEQ ID NO: 7(MY8094-2-80)

TACTATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGC CCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGG CTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATACACCTCAATTACGTACT CCATGGCCTGGTTCCGCCAGGCTCCAGGGAAAGAGCGCGAGGGGGTCGCATTTATT CGTTCCGGTGGTAGTATCACATATTATGCTGACTCCGTGAAGGGCCGATTCACCATC TCCCGTGACGGCCCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAAACCTGA GGACACTGCCATGTACTACTGTGCGGCAGGTCTCCGGGGGTACGATGGTGACTGGT ACGACGGGACTCACTTTCGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAG CGGCCGCATACCCGTACGACGTTCCGGACTACGGTTCCCACCACCATCACCATCACT AGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCT GGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAAT GCTACAGGCGTTGTCGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTT CCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCT GAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACC TATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGA GCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTC ATGTTTCAGAATAATAGGTTCCGAAATAGGCAGGGTGCATTAACTGTTTATACGGGC ACTGTTACTCAAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCA TCAAAAGCCATGTATGACGCTTACTGGAACGGTAAATTCAGAGACTGCGCTTTCCAT TCTGGCTTTAATGAGGATCCATTCGTTTGTGAATATCAGGGCAATCGTCTGACCTGC CTCAACTCCTGTCATGCTGGCGGCGGCTCTGGGGGTGTTCTGGTGCCGCTCTA

SEQ ID NO: 12(MY8094-2-32)

TAATATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGC CCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGCGCAGC CTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCGGCTTCAGTTACAGTG CCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTCGAGTGGGTCTCAACAATT GATAGTCGCGGTGGCGCGACATCGTATGCAGACTCCGTGAAGGGCCGATTCACCAT CTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAAAACTG AGGACACGGCCATGTATTACTGTTCGACGGGGTATAGAGCGTATGACAATAGTCCTC GAGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCATACCCGTACGACGTT CCGGACTACGGTTCCCACCACCATCACCATCACTAGACTGTTGAAAGTTGTTTAGCA AAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGA TCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTCGTTTGTAC TGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGA AAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTG GCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCA ACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATC CTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATAGGTTCCG AAATAGGCAGGGTGCATTAACTGTTTATACGGGCACTGTTACTCAAGGCACTGACCC CGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTATGACGCTTA CTGGAACGGTAATTCAGAGACTGCGCTTTCATTCTGGCTTAATGAGGATCATTCGTT GTGATATCAGGCCATCGCTGACTGCTCACCTCTGTAAGCTGGGGCGGTCTGGTGTGG TCCGGTGGGGCTCTAAGGTGGCGCTCTGAGGTGGGGCTCTGAGGGGGCGTCTGAAG TGGGGCTCTAGGGGGGGTCCGGGGCGCCCCGTCCGGGATTTATAAA

SEQ ID NO: 17(MY8094-2-73)

TATGAAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCC AGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCT GGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACACCTACAGTGGCAACTAC ATAGTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGCAGCTAT TTATCCTTATGCTGGTGGTTATAGCACATACTATGGCAACTTCGTGAAGGGCCGATT CACCATCTCCCGAGACACGGCCAAGAACACGGTGTATCTCCAAATGAACAGCCTGA AACCTGAGGACACTGCCATGTACTACTGTGCGGCAGATAAGCGCGGTATGGGGTCA ATTCTCGCCGTCGAAGGGTATAACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCC TCAGCGGCCGCATACCCGTACGACGTTCCGGACTACGGTTCCCACCACCATCACCAT CACTAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAAC GTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGG AATGCTACAGGCGTTGTCGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGG GTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGT TCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATAC ACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACT GAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACT TTCATGTTTCAGAATAATAGGTTCCGAAATAGGCAGGGTGCATTAACTGTTTATACG GGCACTGTTACTCAAGGCACTGACCCCGTTAAACTTATTACCAGTACACTCCTGTAT CATCAAAGCATGTATGACGCTTACTGGAACGGTAATTCAGAGACTGCGCTTTCATTC TGGCTTTATGAGGATCATTCGTTGTGATATCAGGCCATCGCTGACTGCCCACCTCTGT CATGCTGGCGCGGCTCGGGGT

SEQ ID NO: 22(MY8094-4-39)

GAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCG GCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTCCAGGCTGGCGG GTCTCTGAGACTCTCCTGTGTAGGCTCTGGATACATGTTCAGTACCAACGCCATGAG CTGGGTCCGCCAGGCTCCAGGGAAGGAACTCGAGTGGGTCACAACTATTACTGCTG GTGGTCACACCGGCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAC AACGCCAAGAACACGCTGTATCTTCAATTGAACAGCCTGAAAACTGAGGACACGGC CATGTATTACTGTGCAAATCGGTTCTACTCGAGCGACTATGTCGCCAGGCGGGACTT TGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCATACCCGTA CGACGTTCCGGACTACGGTTCCCACCACCATCACCATCACTAGACTGTTGAAAGTTG TTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAA CTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTCG TTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTA TCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCT GAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACT TATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAAT CCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATA GGTTCCGAAATAGGCAGGGTGCATTAACTGTTTATACGGGCACTGTTACTCAAGGCA CTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAGCCATGTATGA CGCTTACTGGAACGGTAATTCAGAGACTGCGCTTTCATTCTGGCTTAATGAGGATCC ATTCGTT

SEQ ID NO: 27(MY8094-4-42)

TGAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCC GGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAG GGTCTCTGAAACTCTCCTGTGCAGCCTCTGGATTCGTCTTCAGTACCAACGCCATGA GCTGGGTCCGCCAGGCTCCAGGGAAGGAACTCGAGTGGGTCACAACTATTACTGCT GGTGGTCACACCGGCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGA CAACGCCAAGAACACGCTGTATCTTCAATTGAACAGCCTGAAAACTGAGGACACGG CCATGTATTACTGTGCAAATCGGTTCTACTCGAGCGACTATGTCGCCAGGCGGGACT TTGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCATACCCGT ACGACGTTCCGGACTACGGTTCCCACCACCATCACCATCACTAGACTGTTGAAAGTT GTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAA ACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTC GTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCT ATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTC TGAGGGTGGCGGTACTAGACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATAC TTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAA TCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAAT AGGTTCCGAAATAGGCAGGGTGCATTAACTGTTTATACGGGCACTGTTACTCAAGGC ACTGACCCCGTTAAACTTATTACCAGTACACTCCTGTATCATCAAAGCATGTATGAC GCTTACTGGAACGGAAATTCAGAGACTGCGCTTTCATTCTGGCTTTAATGAGGA

SEQ ID NO: 32(MY8094-5-5)

ATGAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGC CGGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGA GGGTCTCTGAGGCTCTCCTGTGCAGCCTCTGAATACAAGACAACCTTCGACACCATG GCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGCAAGTATTCGTAC TCGTAGTGGGCTCACGTACTATGCCGGCTCCGTGAAGGGCCGATTCACCATCTCCCC AGACAACGCCAAGAACACGGTGTCTCTGCAAATGAACGACCTGAAACCTGAGGACA CTGCCATGTACTACTGCGCGACAGGCTTCCGTGGATTTCTGCTCGATACCGGGTGGT CCACTTCGGCCCTGTATGATTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAG CGGCCGCATACCCGTACGACGTTCCGGACTACGGTTCCCACCACCATCACCATCACT AGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCT GGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAAT GCTACAGGCGTTGTCGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTT CCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCT GAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACC TATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGA GCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTC ATGTTTCAGAATAATAGGTTCCGAAATAGGCAGGGTGCATTAACTGTTTATACGGGC ACTGTTACTCAAGGCACTGACCCCGTTAAACTTATTACCAGTACACTCCTGGA

SEQ ID NO: 37(MY8094-5-74)

GTCTATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGC CCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGC CTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCGTCTTCAGTACCAACG CCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGAACTCGAGTGGGTCACAACTATT ACTGCTGGTGGTCACACCGGCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCC AGAGACAACGCCAAGAACACGCTGTATCTTCAATTGAACAGCCTGAAAACTGAGGA CACGGCCATGTATTACTGTGCAAATCGGTTCTACTCGAGCGACTATGTCGCCAGGCG GAACTTTGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCATA CCCGTACGACGTTCCGGACTACGGTTCCCACCACCATCACCATCACTAGACTGTTGA AAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGA CAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGT TGTCGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCT TGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCG GTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCT ATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCG CTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAA TAATAGGTTCCGAAATAGGCAGGGTGCATTAACTGTTTATACGGGCACTGTTACTCA AGGCACTGACCCCGTTAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATG TATGACGCTTACTGGAACGGTAAATTCAGAGACTGCGC

Example 4: Cross reactivity of anti-FAP sdAbs using ELISA

The human FAP or cynomolgus monkey FAP were coated on the microtiter plates and incubated at 37°C. The plates were blocked with 1% BSA at room temperature for 2 hrs. The anti-FAP sdAbs were added to the plates in serial dilutions and incubated at room temperature for 1 hour, followed by incubation with anti-His-HRP antibody at 37°C for another hour for detection. The cells were washed four times with PBS-T between each step. TMB solution and H2SO4 were added to the plates and incubated at room temperature for lOmin. The absorbance at 450 nm was recorded on a Bio-Tek Synergy microplate reader.

The ELISA results in Table 2 demonstrated that anti-FAP sdAbs of the present invention could bind to both human and cynomolgus FAP. Table 2 Summary of the ELISA results for anti-FAP sdAbs

Example 5: Binding kinetics of anti-FAP sdAbs using Biacore 8K

The human FAP-Fc was coupled on a CM5 chip using the standard amine coupling procedure. The anti-FAP sdAbs were diluted into serial dilutions and injected at 30 pl/min in HBS-EP buffer at 25°C with association for 90s and dissociation for 600s. The association and dissociation rates were monitored, and the equilibrium dissociation constant (Kd) was calculated. The chip was regenerated in 1 OmM Glycine for 60s for the next anti-FAP sbAb sample.

The Biacore results in Table 3 demonstrated that anti-FAP sdAbs of the present invention could bind to human FAP with a Kd from pM to nM.

Table 3 Summary of the Biacore 8K results for the anti-FAP sdAbs

Example 6: Inhibitory effect of anti-FAP sdAbs on FAP catalytic activity

The anti-FAP sdAbs and the positive control linagliptin were mixed with FAP-Fc at different dilutions and added to the microtiter black plates. The Z-Gly-Pro-AMC substrate was reconstituted in 40% methanol and added to the mixtures. The plates were read immediately at room temperature using SpectraMax i3x Spectrometer with excitation at 360 nm and emission at 460 nm for 30 mins with 1 min interval between reading.

The results in Table 4 demonstrated that anti-FAP sdAbs of the present invention were binders only and could not inhibit the FAP catalytic activity. Table 4 Inhibitory effect of anti-FAP sdAbs on FAP catalytic activity

Example 7: Epitope binning of the anti-FAP sdAb using ELISA

The anti-FAP sdAbs were tested for competition with each other by using ELISA method. The anti-FAP sdAbs were coated on the microtiter plates and incubated at 4°C overnight. The plates were blocked with 13% BSA at room temperature (RT) for 1 hour. The plates were blocked with 1% BSA at room temperature for 2 hrs. The FAP-Fc and anti-FAP sdAbs were added to the plates and incubated at room temperature for 1 hour, followed by incubation with anti-Fc-HRP antibody at 37° C for another hour for detection. The cells were washed four times with PBS-T between each step. TMB solution and H2SO4 were added to the plates and incubated at RT for 2min. The absorbance at 450 nm was recorded on a Bio-Tek Synergy microplate reader. The competition between anti-FAP sdAbs were calculated based on the change in absorption value.

The epitope binding results in Table 5 demonstrated that anti-FAP sdAbs of the present invention could be classified into two groups:

MY8094-2-32, MY8094-2-80, MY8094-4-39, MY8094-4-42, MY8094-5-39 and MY8094- 5-5 were group 1; and MY8094-2-73 was group 2

NC= Non-Competitive C=Competitive

Example 8: Binding of anti-FAP sdAbs with human cancer cell line The FAP expressing cancer cell line U-87MG was used to validate the binding of anti-FAP sdAbs. The Cells were harvested with a non-enzymatic cell dissociation buffer. Cell suspension containing 2x10 ⁶ cells/ml in PBS was incubated at 4°C in the dark with FcR blocking reagent for 15 min to reduce non-specific binding; followed by incubation with sdAbs (30 min) and Alexa Fluor 488 anti-HA antibody for 20 min. The cells were washed twice with PBS between each step. Flow cytometry analyses were performed on a FACSCalibur flow cytometer.

The FACS results in Fig. 3 demonstrated that anti-FAP sdAbs of the present invention could bind to FAP expressing on cancer cell line.

Example 9: SPECT/CT imaging of anti-FAP sdAbs in human cancer cell line xenograft model

The tricarbonyl kit was reconstituted in sodium pertechnetate and incubated at 25 °C for 20 min. The reconstituted kit was cooled down to room temperature and neutralized to pH7-7.5 with hydrochloric acid. The neutralized [ ^99m Tc]-triaquatricarbonyl technetium(I) was added to the anti-FAP sdAb and incubated at 37°C for Ihr. The [ ^99m Tc] labeled anti-FAP sdAbs was quantified for its radiochemical purity with instant thin layer chromatography (iTLC).

1x10 ⁷ cancer cells (U-87MG) with FAP overexpression were inoculated in the flank at shoulder level of nude mice. The tumour bearing mice were randomly assigned for experiment and anesthetized with isoflurane when the average xenograft size reached 150-200mm ³ . [99mTc] labeled anti-FAP sdAbs (about lOug, 37mbq) was intravenously injected with tail vein. Wholebody SPECT/CT images were acquired at 90-min post-injection with 15 min static SPECT and medium resolution whole body CT.

The result demonstrated that the anti-FAP sdAb of the present invention could effectively accumulate in the FAP overexpression tumour and could be used for FAP targeted cancer diagnosis and efficacy evaluationand develop a new generation of FAP targeted therapy.

All documents mentioned in the present application are hereby incorporated by reference in their entireties as if the disclosures in. In addition, it is to be understood that various modifications and changes may be made by those skilled in the field of the invention after reading the contents., in the form of the appended claims. These equivalent forms also apply for the scope defined by the appended claims.