PL244962B1 - Anti-FOLR1 antibody and its use in medicine - Google Patents

Abstract

The application relates to an antibody that binds to the FOLR1 receptor and exhibits antibody-dependent cellular cytotoxic activity. More specifically, the application relates to an anti-FOLR1 antibody and its conjugates with pharmacologically active compounds. Furthermore, the application relates to pharmaceutical compositions containing such antibodies and their conjugates.

Description

Description of the invention

Technical field

The present invention relates to an antibody that binds to the folate receptor α (hereinafter referred to as FOLR1, Folate Receptor Alpha) and exhibits antibody-dependent cellular cytotoxic activity. More specifically, the invention relates to an anti-FOLRI antibody and its conjugates with pharmacologically active compounds. The invention further relates to pharmaceutical compositions containing such antibodies and their conjugates.

FOLR1 is a GPI-anchored membrane protein with high affinity for folate and has important functions related to cell proliferation or survival (Int J Cancer, 2006. 119(2): p.243-50.). FOLR1 shows a pattern of restricted expression in normal tissues of kidney, lung, intestine, etc. (Anal Biochem, 2005. 338(2): pp. 284-93).

High expression of FOLR1 is observed in various cancers such as ovarian cancer Anal Biochem, 2005. 338(2): pp. 284-93, J Thorac Oncol, 2012. 7(5): pp. 833-840) J Thorac Cardiovasc Surg , 2001. 121(2): p. 225-33), kidney cancer (Anal Biochem, 2005. 338(2): p. 284-93), lung cancer (Anal Biochem, 2005. 338(2): p. 284-93, breast cancer (Anal Biochem, 2005. 338(2): pp. 284-93).

In particular, the expression level of FOLR1 has been reported to be associated with the grade, progression and prognosis of ovarian cancer (Int J Cancer, 1997. 74(2): p. 193-8, Int J Cancer, 1998. 79(2): p. .121-6). Furthermore, it was also reported that soluble FOLR1 was significantly increased in the serum of ovarian cancer patients compared to the serum of healthy donors (PLoS One, 2009. 4(7): p.e6292). Thus, FOLR1 is a promising target molecule for the treatment of these types of cancer.

LK26 is known as a mouse monoclonal antibody against FOLR1 (EP 0197435). LK26 was humanized by changing the CDR for administration to a patient as an antibody for the treatment of cancer (US 5,952,484). However, his affinity has been greatly reduced by humanization.

There is a known antibody MORAb-003 that has equivalent affinity for FOLR1 to the LK26 antibody, based on the humanized LK26 antibody (Cancer Immun, 2007. 7: p. 6). The in vitro antitumor activity of MORAb-003 is believed to be attributed to ADCC activity and CDC activity.

On the other hand, there is a report that MORAb-003 has no activity to inhibit the binding of folate, 5-methyltetrahydrofolate (5-MTHF), or pemetrexed in FOLR1-positive cells, and the antibody itself does not inhibit the proliferation of FOLR1-positive cells (Cancer Chemother Pharmacol, 2012 .70(1): p.113-20).

A phase I clinical trial showed that MORAb-003 is safe and tolerable at doses ranging from 12.5 to 400 mg/m ² (Clin Cancer Res, 2010. 16(21): p. 5288-95). Moreover, a Phase II clinical trial showed that MORAb-003 in combination with paclitaxel and carboplatin demonstrated significant antitumor activity in patients with recurrent platinum-sensitive ovarian cancer (J Clin Oncol, 2010, 28(15): 5001).

However, the clinical trial in patients with recurrent platinum-resistant ovarian cancer was discontinued due to the possibility of failure to meet the endpoint of improvement in prespecified statistical criteria (progression-free survival or overall survival) at the interim analysis.

An example of an anti-cancer agent targeting FOLR1 would be EC-145, which is produced by conjugating the anti-cancer agent to folate, in addition to the above anti-FOLR1 antibody. The folate-conjugated anticancer agent demonstrates its efficacy based on the folate uptake properties of FOLR1. Therefore, it is suggested that the folate-conjugated anticancer agent will be ineffective in cancer cells with low folate uptake activity, although FOLR1 is expressed in the body's cancer cells.

Despite continuous progress in the treatment of solid cancers and improved prognosis, there has been no significant improvement in the prognosis of ovarian cancer since the introduction of platinum-based therapy in 1980 (NatRev Cancer, 2011. 11(10): p. 719-25), and ovarian cancer is still the leading cause of cancer death in women. The reason is that ovarian cancer becomes resistant to platinum and, as a result, recurrent ovarian cancer is incurable.

This means that the challenges in treating ovarian cancer are prolonging the period before recurrence after response to chemotherapy with a platinum or similar agent, preventing ovarian cancer from becoming resistant to a platinum agent, and establishing therapy for platinum-resistant ovarian cancer.

An additional problem in antibody therapy is the molecular weight of a conventional monoclonal antibody (~150 kD), which is too large to allow the antibody to penetrate the tissue, resulting in a lower effective concentration in the tumor region and an insufficient therapeutic effect.

Moreover, a number of facts, such as the long development cycle, high production costs and lack of stability of fully humanized traditional antibodies, limit their use and popularity in clinical practice. It is also problematic to validate large-scale assembly processes of antibody fragments containing light and heavy chains, and therefore laboratory solutions targeting FOLR1 are difficult to translate to production scale and therefore unavailable in therapy.

Thus, there is still a lack of satisfactory antibody constructs or fragments thereof directed against FOLR1 that could be used in therapy. Therefore, an urgent need in the field is to develop new specific constructs that are effective against cancers associated with FOLR1 expression.

Muyldermans, S. (2013) Annu. Fr. Biochem. 82, 775-797 discloses nanobodies: single-domain antibodies and highlights its advantages, including their favorable biochemical and economic properties; size affinity, specificity, stability and production costs. Single-domain antibodies (sdAbs) differ from conventional 4-chain antibodies in that they have a single variable domain of a monomeric antibody. For example, camelids and sharks produce sdAbs called heavy chain-only antibodies (HcAbs), which naturally lack light chains. The antigen-binding fragment in each arm of the camelid heavy chain-only antibody has a single heavy chain variable domain (VHH) that can have high affinity for antigen without the help of the light chain. The camelid VHH is known to be the smallest functional antigen-binding fragment with a molecular weight of approximately 15 kD. Such VHH molecules can be used independently, e.g. in various immunological tests (instead of sc Fv, IgG) or serve independently or as a carrier of the active substance. However, one type of antigen-binding fragment may not be sufficient to provide effective therapy for FOLR1-related cancers. In turn, dimerization of the VHH chains themselves reduces the binding efficiency of the target site.

The present invention addresses the above problems by providing two different VHH fragments directed against FOLR1 that exhibit anticancer activity against cell lines in vitro and in vivo. Such VHH fragments allow the reconstitution of full-length antibodies using the constant fragment of the heavy chain of human IgG antibodies. The antibody reconstituted in this way retains the properties and binding strength of the antigen, and the additionally introduced constant chain allows for the efficient preparation of heterodimeric antibodies. Furthermore, the present invention provides a method for producing an anti-FOLR1 antibody, conjugating it with pharmacologically active molecules, and a pharmaceutical composition containing such an antibody or a conjugate based thereon.

More specifically, the subject of the invention are VHH sequences that are a modified heavy chain derived from antibodies of animals from the camelidae family, which includes, among others, camels and llamas (such as Lama gama). The antibodies of these animals have two types of variable chains: VH and VHH, characteristic of the IgG2 and IgG3 antibody subclasses of this family of animals. They are characterized by the ability to bind antigen. Compared to the classic heavy chain variable fragment, they have an extended CDR3 domain and an additional disulfide bridge that stabilizes the molecule. VHH fragments have numerous advantages over the commonly used single-chain fragment variable-scFv. First of all, the procedure and ease of cloning from various libraries: native, synthetic or libraries obtained after immunization of llamas are very simplified. VHH is very easy to produce in bacteria: in large quantities and in a soluble form, which facilitates purification and minimizes production costs. VHH molecules are also characterized by high stability, which makes it easier to work with them, as well as high and specific binding to antigens. VHH molecules can be used independently, e.g. in various immunological tests (instead of scFv, IgG) or serve as a carrier of an active substance, which is being increasingly tested, e.g. in cancer therapy. Selected VHH fragments also allow the reconstruction of full-length antibodies using, for example, a constant chain from human IgG antibodies, and therefore can serve as a starting point for the preparation of another class of antibody molecules. The subject of the invention is to obtain antibodies that bind to the FOLR1 receptor in such a way that the first VHH fragment binding to the FOLR1 receptor is attached to one of the arms of the constant fragment of the heavy chain of human IgG antibodies, and the second VHH fragment binding to the second arm of the said heavy chain is attached to the second arm of the heavy chain. the FOLR1 receptor, wherein the mentioned VHH fragments are different from each other. The attached constant fragment of the heavy chain of human IgG antibodies has a mutation introduced that allows for the efficient preparation of heterodimeric antibodies. The obtained antibodies constitute a carrier to which the cytostatic molecule is then conjugated using chemical methods. The obtained immunoconjugate is used in the therapy of cancers characterized by overexpression of the FOLR1 receptor. This type of overexpression is characteristic of, among others, for ovarian cancer.

In a first aspect, the present invention provides:

a first anti-FOLR1 VHH chain, comprising a complementarity determining region (CDR) of the VHH chain consisting of CDR1 as shown in SEQ ID NO.: 1, CDR2 as shown in SEQ ID NO.: 2 and CDR3 as shown in SEQ ID NO.: 3.

In a second aspect, the present invention provides a second anti-FOLR1 VHH chain comprising a complementarity determining region (CDR) of the VHH chain consisting of CDR1 as shown in SEQ ID NO: 4, CDR2 as shown in SEQ ID NO: 5 and CDR3 as shown in in SEQ ID NO.: 6.

Said CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain, and the binding of said first and second anti-FOLR1 VHH chains can block the binding of ligands to FOLR1 and induce cellular cytotoxicity.

A third aspect of the present invention provides a first anti-FOLR1 VHH chain, said VHH chain comprising a framework region (FR) and a complementarity determining region (CDR) according to the first aspect, said framework region consisting of FR1 as set forth in SEQ ID NO:7 , FR2 as shown in SEQ ID NO: 8, FR3 as shown in SEQ ID NO: 9 and FR4 as shown in SEQ ID NO: 11.

A fourth aspect of the present invention provides a second anti-FOLR1 VHH chain, said VHH chain comprising a framework region (FR) and a complementarity determining region (CDR) according to the second aspect, said framework region consisting of FR1 as set forth in SEQ ID NO:7 , FR2 as shown in SEQ ID NO: 8, FR3 as shown in SEQ ID NO: 10 and FR4 as shown in SEQ ID NO: 11.

In a preferred embodiment, the first anti-FOLR1 VHH chain has the sequence as set forth in SEQ ID NO.: 12.

In a preferred embodiment, the second anti-FOLR1 VHH chain has the sequence as set forth in SEQ ID NO.: 13.

A fifth aspect of the present invention provides a first anti-FOLR1 antibody fragment that is directed against FOLR1 and comprises a first VHH chain as set forth in the amino acid sequence of SEQ ID NO: 12, wherein said first VHH chain is operably linked to a first Fc constant heavy chain fragment human IgG as set forth in the amino acid sequence of SEQ ID NO: 14 and said first antibody fragment is as set forth in SEQ ID No: 16.

A sixth aspect of the present invention provides a second anti-FOLR1 antibody fragment that is directed against FOLR1 and comprises a second VHH chain as set forth in the amino acid sequence of SEQ ID NO: 13, wherein said second VHH chain is operably linked to a second Fc constant heavy chain fragment human IgG as set forth in the amino acid sequence of SEQ ID NO: 15 and said second antibody fragment is as set forth in SEQ ID No: 17.

The seventh aspect of the present invention provides an anti-FOLR1 antibody composed of the fragments of the fifth and sixth aspects of the invention folded into a heterodimer. An anti-FOLR1 antibody heterodimer consists of the first antibody fragment represented by SEQ ID NO: 16 and the second antibody fragment represented by SEQ ID NO: 17, linked to form a heterodimer by mutations introduced in the Fc region.

The eighth aspect of the present invention provides polynucleotides, said polynucleotides encoding:

proteins of a first anti-FOLR1 antibody fragment according to the fifth aspect of the present invention as set forth in SEQ ID NO:21 and a second anti-FOLR1 antibody fragment according to the sixth aspect of the present invention as set forth in SEQ ID NO:22;

a protein of a first human IgG Fc heavy chain constant fragment as set forth in the amino acid sequence of SEQ ID NO: 19 and a second human IgG heavy chain Fc constant fragment as set forth in the amino acid sequence of SEQ ID NO: 20;

anti-FOLR1 first VHH chain as shown in SEQ ID NO: 23;

the second anti-FOLR1 VHH chain as shown in SEQ ID NO: 24.

In one preferred embodiment, said polynucleotides according to the eighth aspect of the invention have a nucleotide sequence as set forth in SEQ ID NO: 21 and SEQ ID NO: 22.

In another preferred embodiment, said polynucleotides are DNA sequences.

The ninth aspect of the present invention provides an expression vector, which expression vector comprises polynucleotides according to the eighth aspect of the present invention, in particular polynucleotides according to the eighth aspect of the invention that have a nucleotide sequence as set forth in SEQ ID NO.: 21 and SEQ ID NO.: 22.

A tenth aspect of the present invention provides a host cell, and said host cell comprises an expression vector according to the ninth aspect of the present invention or polynucleotides according to the eighth aspect of the present invention integrated into the genome of the host cell.

In one preferred embodiment, said host cell is an E. coli cell or a mammalian cell.

An eleventh aspect of the present invention provides a method of producing anti-FOLR1 antibodies comprising the steps of:

(a) culturing said host cell as defined in the tenth aspect of the present invention under conditions suitable for producing antibodies, thereby obtaining a culture comprising said anti-FOLR1 antibodies; and (b) isolating or recovering said anti-FOLR1 antibodies from said culture.

In a preferred embodiment, said anti-FOLR1 antibody has an amino acid sequence defined by the sequence of the first heterodimer chain of SEQ ID NO: 16 and the second heterodimer chain of SEQ ID NO: 17.

A twelfth aspect of the present invention provides an immunoconjugate, said immunoconjugate comprising:

(a) an anti-FOLR1 antibody according to the seventh aspect of the present invention; and (b) a conjugated moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide and an enzyme.

In another preferred embodiment, said conjugated moiety is a drug or toxin.

In another preferred embodiment, said conjugate portion is a detectable marker.

In another preferred embodiment, said 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 (e.g. IL-2 ). etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles, virus particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g. cisplatin, maytansinoids, auristatin and its derivatives such as Monomethylauristatin E (MMAE)) or any forms of nanoparticles etc. that produce detectable products.

The thirteenth aspect of the invention provides the anti-FOLR1 antibody of the seventh aspect of the present invention for use as (a) an agent for detecting a FOLR1 molecule; or (b) a drug for the treatment of cancer.

In another preferred embodiment, said detection comprises detection by flow cytometry or cellular immunofluorescence.

A fourteenth aspect of the invention provides a pharmaceutical composition comprising:

(i) an anti-FOLR1 antibody according to the seventh aspect of the present invention or an immunoconjugate according to the twelfth aspect of the present invention; and (ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, said pharmaceutical composition is in an injectable form.

In another preferred embodiment, said pharmaceutical composition is for use in the treatment of cancer, and said cancer is selected from the group consisting of ovarian cancer, kidney cancer, lung cancer, breast cancer.

The fifteenth aspect of the present disclosure provides one or more uses of the anti-FOLR1 antibody according to the seventh aspect of the present invention or the immunoconjugate according to the twelfth aspect of the present invention:

(i) for detecting the human FOLR1 molecule;

(ii) for flow cytometry assay;

(iii) for detection of cellular immunofluorescence;

(iv) for the treatment of cancer;

(v) for diagnosing cancer.

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

The sixteenth aspect of the present invention provides for the use of the anti-FOLR1 antibodies according to the seventh aspect of the present invention and/or the immunoconjugate according to the twelfth aspect of the present invention to prepare an agent, detection plate or kit; wherein said detection means, plate or kit is used to detect the FOLR1 protein in the test sample. Further disclosed is the use of an anti-FOLR1 antibody according to the seventh aspect of the present invention and/or an immunoconjugate according to the twelfth aspect of the present invention in the preparation of a medicament, wherein the medicament is used to treat or prevent cancers expressing FOLR1 (i.e., FOLR1-positive).

In another preferred embodiment, said cancer includes ovarian cancer, kidney cancer, lung cancer, breast cancer, most preferably ovarian cancer.

Brief description of the figures

Figure 1 shows the results of anti-FOLR1 VHH domain affinity studies as shown in SEQ ID NO: 12.

Figure 2 shows the results of the anti-FOLR1 domain affinity studies as shown in SEQ ID NO: 13.

Detailed description

Anti-FOLR1 antibodies and conjugates based thereon according to the invention can effectively block the interaction between FOLR1 and its ligands. BiaCore T200 analysis shows that anti-FOLR1 antibodies have high affinity, excellent stability and significant tumor-suppressive activity.

As used herein, the terms "antibodies of the invention", "anti-FOLR1 antibodies of the invention" and "FOLR1 antibodies of the present invention" are interchangeable and refer to antibodies that specifically recognize and bind to FOLR1 L1 (including human FOLR1). More preferred said anti-FOLR1 antibodies according to the seventh aspect of the present invention.

As used herein, the term "antibody" means a heterodimeric antibody that binds to FOLR1 receptors, in which a first FOLR1 receptor-binding VHH fragment is attached to the first arm of the human IgG heavy chain constant fragment and a second FOLR1 receptor-binding VHH fragment is attached to the second arm of the human IgG heavy chain. fragment of VHH binding to the FOLR1 receptor, wherein the first and second fragments of the heavy chain of human IgG antibodies and the first and second fragments of VHH binding to the FOLR1 receptor are different from each other, and the first and second fragments of the heavy chain of human IgG antibodies have introduced mutations allowing for efficiently obtaining said heterodimeric antibody, i.e., forming and folding into heterodimers upon expression.

As used herein, the term VHH refers to the variable region of the heavy chain of an antibody. It is the smallest antigen-binding fragment with full function.

As used herein, the term "variable" refers to the fact that certain portions of the variable region in antibodies differ in sequence, which creates binding and specificity of different specific antibodies to their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved part of the variable region is called the framework region (FR). Each of the natural heavy and light chain variable regions contains four FR regions that are essentially in a β-fold configuration, linked by three CDRs that form a connecting loop and, in some cases, may form a partially β-folded structure. The CDR regions in each chain are closely adjacent to the other FR regions and form the antibody antigen binding site with the CDR regions of the second chain (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pages 647-669. (1991)) . Constant regions are not directly involved in antibody-antigen binding, but have various effects or functions, for example engaging in antibody-dependent antibody cytotoxicity.

As known to 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 antibodies or fragments thereof of the present invention. The disclosure also includes a cell surface marker or antigen that binds to said anti-FOLR1 antibody or fragment thereof.

As used herein, the terms "heavy chain variable region" and "Vh" may be used interchangeably.

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

The heavy chain variable region of said antibody contains 3 complementarity determining regions: CDR1, CDR2 and CDR3.

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

In accordance with the present invention, the terms "antibody 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 a polypeptide with a heavy chain variable region that specifically binds to the protein FOLR1. They may or may not contain starting methionine.

The invention also provides other proteins or fusion expression products containing the antibodies of the invention. In particular, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, as long as the variable regions are identical or at least 90%, preferably at least 95% identical with the heavy chain of the antibody of the present invention.

Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the heavy chain variable region, referred to as variable regions (CDRs), and the segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in binding reactions. Said CDRs form a loop structure in which the β-sheets formed by the FRs between them 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 antibody. The amino acid sequences of the same type of antibodies can be compared to determine which amino acids constitute the FR or CDR regions.

The variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in antigen binding. Therefore, the present disclosure includes those molecules that have an antibody heavy chain variable region with CDRs, provided that their CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) identical to the CDRs identified herein.

The present invention includes not only intact antibodies, but also fragment(s) of an immunologically active antibody or a fusion protein formed from an antibody with other sequences. Therefore, the present invention also includes antibody fragments, derivatives and analogues, such as the single chain heterodimeric antibodies of the fifth and sixth aspects of the invention.

As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the same biological function or activity of an antibody of the invention. The polypeptide fragments, derivatives or analogues of the invention may be (i) polypeptides having substituted one or more conserved or non-conservative amino acid residues (preferably non-conservative amino acid residues). Such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide containing a substituent group at one or more amino acid residues; or (iii) a polypeptide formed by fusing the 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 a polypeptide sequence (e.g., a leader or secretory sequence or sequence used to purify the polypeptide, or a proprotein sequence or fusion protein formed with a 6xHis tag). As used herein, these fragments, derivatives and analogues are within the scope of one of ordinary skill in the art.

An antibody of the present invention relates to a polypeptide comprising the above CDR regions having FOLR1 protein binding activity. The term also includes variant forms of polypeptides containing the above CDR regions that perform the same function as the antibodies of the invention. These changes include, but are not limited to, deletion insertions and/or substitutions of one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids and the addition of one or more (usually less than 20, preferably less than 10, and more preferably less than 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution of amino acids with analogous or similar properties does not usually alter the function of the protein. As another example, adding one or more amino acids at the C-terminus and/or N-terminus does not usually change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variant polypeptide forms include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA capable of hybridizing to DNA encoding an antibody of the present invention under stringent or mild conditions, and polypeptides or proteins obtained using an antiserum against the antibodies according to the invention.

The invention also provides other polypeptides, such as a fusion protein containing antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the antibodies of the invention. Typically, the fragment comprises at least about 50 contiguous amino acids of the antibody 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 an antibody of the present invention" refers to 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 amino acids having properties analogous or similar to the amino acid sequence of the antibody of the present invention invention. These conserved polypeptide variants are preferably produced according to the amino acid substitutions in Table 1.

PL 244962 Β1

Table 1

The rest is original {Example substitution I Favorable substitution Ala (A) jVal, Leu, Ile j Val Arg (R) (Lys; Gin; Asn Lys Asn (N) |dn; Ilis; Lys; Arg i Gin Asp (D) |g1u l Glu Cys (C) | Cheese and Cheese Gin (Q) I Asn l Asn Glu (E) (Asp f Asp Gly(G) jPro, Ala Alas His (H) ¥ | Asn; Gin; Lys; Arg Arg He (I) (Leu; Val; Met; Ala; Phe Leu Leu (L) [how much; Val; Underworld; Ala; Phew How much Lys (K) jArg; Gin; Asn Arg Met (M) |Leu; Phe; How much Leu Phe (F) |Leu; Val; How much; Ala; Tyr Leu Pro (P) j Ala Alas Cheese (S) |ίΤιγ Thr Thr (T) | Cheese Cheese Trp (W) | Tyre; Phew Tyr Tyr (Y) |Trp, Phe; Thr; Cheese Phew Val (V) jlle; Leu; Underworld; Phe; Alas Leu

The present invention also provides a polynucleotide molecule encoding the above antibody or a fragment or fusion protein thereof. The polynucleotides of the invention may be in the form of DNA or RNA. Forms of DNA 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 mature polypeptides of the invention include: coding sequences only encoding the mature polypeptide; mature polypeptide coding sequences and various additional coding sequences; coding sequences (and optional additional coding sequences) and non-coding sequences of the mature polypeptide.

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

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

The full-length nucleotide sequence of an antibody of the present invention or a fragment thereof may generally be obtained by PCR amplification, recombinant method, or artificial synthesis method. One possible method is to synthesize related sequences using synthetic methods, especially when the fragment length is short. Generally, a long sequence of fragments can be obtained by first synthesizing many small fragments and then combining them. Additionally, a heavy chain coding sequence and an expression tag (e.g., 6xHis) may be linked together to form a fusion protein.

Once the appropriate sequences are obtained, the sequences in question can be obtained on a large scale using recombinant methods. Typically, sequences can be obtained by cloning them into a vector, transferring them into cells, and then isolating the sequences from proliferating host cells by conventional methods. Biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in isolated form.

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

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

The host cell may 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, Drosophila S2 or Sf9 insect cells, CHO animal cells, COS7, 293 cells and the like.

Transformation of a host cell with recombinant DNA can be accomplished using conventional techniques well known to those of skill in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with CaCl2. The procedures used are well known in the art. Another method is to use MgCl2. Electroporation can also be used if necessary. 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 resulting transformants can be grown in a conventional manner to express the polypeptide encoded by the gene of the present invention. Depending on the host cells used, the culture medium can be selected from various conventional media. The culture is carried out under conditions suitable for the growth of host cells. After the host cells have been grown to the appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature change 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, taking advantage of its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultracentrifugation, molecular sieve chromatography (gel filtration), ion exchange chromatography, high performance liquid chromatography (HPLC), and various other chromatography techniques liquid and their combinations.

The antibodies of the invention may be used as such or in combination with or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying 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 may be bound or conjugated to the antibodies 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; 5. Virus particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (for example, DT diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutic agents (for example cisplatin, maytansinoids, auristatin and its derivatives) etc., preferably Monomethylauristatin E (MMAE).

Pharmaceutical composition

The invention also provides a composition. Said composition is a pharmaceutical composition containing the above antibody or an active fragment thereof or a fusion protein or an immunoconjugate formed on the basis thereof, and a pharmaceutically acceptable carrier. Generally, these materials can be formulated in non-toxic, inert and pharmaceutically acceptable aqueous vehicles in which the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulation material and the condition, who is to be treated. The formulated pharmaceutical compositions may 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 for the treatment of cancer. In addition, other therapeutic agents may be used concomitantly.

The pharmaceutical composition of the present invention contains a safe and effective amount (e.g. 0.001-99 wt.%, preferably 0.01-90 wt.%, and more preferably 0.1-80 wt.%) of the above-mentioned antibodies 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 combinations thereof. The composition of the drug should be appropriate to the method of administration. The pharmaceutical composition of the present invention may be prepared by injection, for example by a conventional method using saline or an aqueous solution containing glucose and another adjuvant. Pharmaceutical compositions such as injections and solutions are preferably prepared 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. Furthermore, the polypeptides of the invention may also be used with other therapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 5-10 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, to determine specific dosages, factors such as the route of administration and the patient's medical condition must be taken into account, all of which are within the skill of qualified physicians.

The antibodies of the invention are distinguished from the state of the art in the field of anti-FOLR1 antibodies in that (a) the antibodies of the invention are anti-FOLR1 proteins with high human specificity and correct spatial structure;

(b) the antibodies of the invention have high affinity; and (c) the antibodies of the invention are produced by a simple method.

The present invention is further described in connection with specific embodiments. It is to be understood that these examples serve only to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods that do not specify specific conditions in the examples below are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or in accordance with the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight and parts by weight.

Embodiments of the invention

Example 1: Expression and purification of the FOLR1 receptor in a mammalian expression system

Cloning and purification of plasmids

The nucleotide sequence encoding the FOLR1 receptor was optimized for efficient expression in human cells using the art-known Gene Art algorithm (Thermo Scientific). The sequences of the above-mentioned receptor fused to the Fc fragment of the antibody (SEQ ID NO: 18) were cloned into the pSF2 plasmid. The sequence of the FOLR1 receptor according to UniProtKB/Swiss-Prot was used: P15328.3 amino acids 25-234 corresponding to the mature FOLR1 receptor chain.

The correctness of the cloned nucleotide sequences encoding extracellular receptor fragments into the appropriate plasmids was confirmed by sequencing. The appropriate constructs were propagated in E. coli strain DH5a and purified using MachereyNagel™ NucleoBond™ Xtra Maxi Plus EF kit according to the manufacturer's instructions. The final preparations were additionally sterile filtered through a 0.22 pm filter.

Transfection

Expi293F™ cells were cultured under standard conditions according to the supplier's protocol (37°C, 8% CO2, 100 rpm). For transfection, 7.5 x 107 cells in 25.5 ml of Expi293F™ Expression Medium (small scale) or 14.5 x ¹⁰⁸ cells in 425 ml of medium for large scale production were used, respectively. Cells were cultured in 125 ml sterile, disposable Erlenmeyer flasks. Lipid-DNA complexes per 30 ml or 500 ml of transfection, respectively, were obtained as follows:

a) 30 pg or 500 pg of plasmid, respectively, was diluted with Opti-MEM® I Reduced Serum Medium in a final volume of 1.5 ml or 25 ml, respectively, mixed gently and incubated for 5 min.

b) 80 pl or 1350 pl ExpiFectamine™ 293 Reagent was diluted with OptiMEM™ I medium in a final volume of 1.5 ml or 25 ml, respectively, mixed gently and incubated for 5 min.

After 5 min of incubation, the diluted DNA was added to the diluted ExpiFectamine™ 293 Reagent, mixed gently and incubated further to form DNA-ExpiFectamine™ 293 Reagent complexes. The prepared complexes were added to the cells and incubated for 18 hours in the above-mentioned conditions with shaking at 140 rpm. After this time, transfection enhancers were added according to the manufacturer's protocol. In order to check the efficiency of protein production, 200 pl samples were taken from the cultures on days 2, 3, 4 and 5. The samples were centrifuged to separate the cells from the medium. Both fractions were frozen at -20°C for further analysis. To verify the presence of protein, individual fractions were analyzed using SDS-PAGE electrophoresis. Protein samples were separated on 4-20% Mini-PROTEAN® TGX Stain-Free™ Protein Gel and stained.

Protein purification

The FOLR1 receptor overexpressed in Expi293F™ cells was then purified.

Protein purification on a small scale

Small-scale purification of the overexpressed receptor was performed by affinity chromatography on a protein A column according to the column manufacturer's instructions. Purification of proteins from the post-culture medium was performed at 4°C. The protein eluted with TEA (triethylamine) was neutralized with a pH 8.0 buffer and concentrated on an Amicon Ultra-15 Centrifugal Filter Unit with a 10 kDa membrane (Merck), and then the buffer was exchanged by dialysis against 50 mM TrisHCl, 300 mM NaCl and 10% glycerol, pH 7.5.

Large-scale protein purification

Large-scale purification of the overexpressed receptor was carried out in two steps: using affinity chromatography on a HisTrap™ Ni NTA 5 ml column (GE Healthcare), and then, as before, using affinity for protein A. After binding to the column, the proteins were washed with a buffer containing: 50 mM TrisHCl, pH 7.5, 300 mM NaCl, 30 mM imidazole. Proteins bound to the column were eluted with the same buffer containing a gradient increasing concentration of imidazole (50 - 300 mM). The purified protein was concentrated on an Amicon Ultra-15 Centrifugal Filter Unit with a 10 kDa membrane (Merck) and then the buffer was exchanged by dialysis against 50 mM TrisHCl, 300 mM NaCl and 10% glycerol, pH 7.5. The pre-purified protein was finally purified on protein A as previously described. Concentration was measured using the Bradford method.

Cutting off the Fc fragment and obtaining a pure receptor

In order to remove the Fc fragment from the fusion protein and obtain pure, soluble receptors, TEV digestion was used. The reaction mixture was loaded onto a protein A column and then onto an SEC column or cleaned on a nickel column to remove the digested Fc fragment and undigested full-length fusion protein. The concentration of the obtained receptor was measured by the Bradford method and, after deglycosylation, sent for LC-ESI-MS analysis to confirm the mass.

Immunization of llamas Llama (Lama gama) were immunized with purified protein that did not contain the Fc-FOLR1 fragment. The animals were immunized 4 times, on days 0, 14, 28 and 35. One week after the last immunization, blood was collected from the animals and RNA was isolated.

The post-vaccination response was tested using the enzyme-linked immunosorbent assay ELISA. For this purpose, a 96-well plate was coated with the appropriate antigen and incubated overnight at 4°C. To minimize non-specific binding and block vacant sites, the plate was incubated with 4% milk and then serial dilutions of sera were applied. After an hour, unbound antibodies were washed away, and bound VHH was detected with anti-VHH antibodies, and then anti-rabbit antibodies labeled with horseradish peroxidase were added. The binding of VHH to the appropriate antigen was quantified by generating a color reaction of o-phenylenediamine in the presence of H2O2 read at a wavelength of 490 nm.

Example 2: Construction of an anti-FOLRI antibody variable fragment library

Preparation of a phage library

Blood samples for RNA isolation were collected on the 43rd day after the first immunization of the llamas. To check the integrity of the RNA, the samples were loaded on an agarose gel. 40 μg of purified RNA was transcribed into cDNA using Invitrogen reverse transcriptase. The synthesized cDNA was purified using a PCR clean-up kit (Macherey-Nagel). The heavy chains (normal and heavy) of the antibodies were amplified using primers binding to the leader sequence and the CH2 region. Analysis of the reaction on a 1% agarose gel confirmed the presence of 2 bands of approximately 700 bp and approximately 900 bp representing both types of heavy chains: VHH and VH. The corresponding band (700 bp) was then excised from the agarose gel and purified. 80 hives were used as a matrix in the so-called internal PCR. The PCR product was purified and digested with Sfil and then BstEll enzyme to obtain a 400-bp product visible on the agarose gel. The obtained band was cut out from the gel again, purified and then cloned into the pUR8100 phagemid vector and transformed into E. coli bacteria of the TG1 strain. TG1 transformants were titrated using 10-fold dilutions and spotted on LB agar plates supplemented with ampicillin (100 μg/ml) and 2% glucose. The titer of the obtained phage library was calculated taking into account its volume, the number of colonies obtained and dilution. The size of the obtained libraries for all tested receptors ranged between 1.44 x 10 ⁸ and 4.8 x 10 ⁸ . Each of the prepared libraries was checked to confirm the frequency of insertions. PCR analysis of 24 clones from each of them confirmed the presence of inserts in all tested clones. Only one of the two libraries prepared for the FOLR1 receptor had an insert frequency >90%.

PL 244962 Β1

Selection of VHHs binding to the F0LR1 receptor using the Phage Display technique

E. coli bacteria of the TG1 strain containing the appropriate library were diluted to ODeoo = 0.05 in 2xTY medium containing 2% glucose and 100 μg/ml ampicillin and cultured for 2 h at 37°C to OD6oo0.5. Then, 7 ml of the culture was infected with the helper phage VCS M13 using MOI (multiplicity of infection) = 100 for 30 min at 37°C. The bacteria were then pelleted and suspended in fresh 2xTY medium supplemented with ampicillin (100 μg/ml) and kanamycin (25 μg/ml) and cultured overnight at 37°C with shaking. The multiplied phages were then precipitated from the supernatant with PEG-NaCL solution. Phage titers were calculated by infecting TG1 bacteria with serial dilutions of phages.

Example 3: Screening, phage selection and verification of anti-FOLR1 VHH

For the analyzed library, a MaxiSorp plate was coated and incubated overnight with the appropriate FOLR1-Fc2 antigen at concentrations of 5 μg/ml and 0.5 μg/ml or PBS as a negative control. After washing away unbound antigens, the plate was blocked with a 4% skimmed milk solution. At the same time, 20 μΙ of precipitated phages (~10 ¹¹ phages constituting over 1000 times the diversity of the libraries) were initially blocked by 30 min incubation with a 2% skimmed milk solution. The prepared phages were incubated for 2 h with the appropriate antigen coated on the plate. After extensive washing with PBST solution, the bound phages were eluted with 0.1 M TEA buffer and immediately neutralized with 1 M TRIS/HCl pH 7.5. The eluted phages were titrated by infecting serial dilutions with TG1 bacteria and then inoculating on a plate supplemented with 2% glucose and ampicillin (100 μg/ml).

The phages obtained during selection were used to infect TG1 bacteria and protected at -80°C as glycerol slopes. At the same time, eluates from wells coated with a higher concentration of antigens were multiplied for the second round of selection. An overnight culture of phage-infected TG1 was diluted 100-fold in 5 ml of fresh 2xYT medium supplemented with 2% glucose and ampicillin (100 μg/ml) and grown to logarithmic phase. Then, 1 μΙ of helper phages were added and incubated for 30 min at 37°C. The multiplied phages were precipitated with PEG-NaCl solution.

μ obtained in the first round of selection and then multiplied phages were used for the second round of selection, this time using a plate coated with 3 antigen concentrations: 5 μg/ml, 0.5 μg/ml and 0.05 μg/ml. Then the procedure steps were the same as in round one.

Similarly, the 3rd round of phage selection was also performed. The obtained clones, obtained in the periplasmic fraction, were tested in the ELISA test. For this purpose, single clones were seeded in duplicate in a 96-well plate containing 1 ml of 2xYT medium with 0.1% glucose ampicillin (100 μg/ml) and cultured for 3 h at 37°C. Then, 1 mM IPTG was added and the culture was continued overnight at room temperature. In the morning, the culture was pelleted, suspended in PBS, frozen and thawed to isolate the periplasmic fraction, and centrifuged to remove membranes and cellular debris.

To confirm the binding of VHH periplasmic fractions to a given receptor, the MaxiSorp plate was coated overnight with 100 ng/ml of a given antigen, blocked with 4% skim milk, and then 10 μΙ of periplasmic fractions containing individual monoclonal VHH were added to the prepared plate and incubated for 1 h with 1% skimmed milk. Bound VHH was then detected with rabbit anti-VHH and then peroxidase-conjugated anti-rabbit antibodies. VHH binding was quantified by a colorimetric OPD reaction in the presence of H2O2 and measured at 490 nm.

Tabk I. Purtfied VHlii dii ećtid Ag.uust JoLR

V«Tof Tag lot CF MW Cortfc. Conc[μΜ$ Ae?wu<iP iwrreber IM _____________________........._

Arn-ISŁ AFR4AS...... pMEK222 FLAG-Hb 'hag-hF™ 180906Lrfł TSSócGr' 'lłU 14741 1529s 1.294 , “Ϊ6Ϊ4 i· ⁸ ®· YmY «MB I Ϊ5Ϊ27' AFR-1D9 fiMEK222 FLAG-His lSD90£vft 1S57 1473B 4.132 2.S8S | 181.47 ΐβ.13 .. AFR-1O2 PMEK222 FLAG-Hb 180906 LvR 1.905 15055 5,786 2,900 ] 192.64 : 0.18

Positive clones were sequenced to confirm their diversity. For each antigen, at least 10 different sequences belonging to 2 different groups (germline families) were obtained: KQREL or KEREF.

From the obtained FOLR1-binding VHH sequences, the following FOLR1-binding VHHs were selected for further analysis:

PL 244962 Β1

AFR-1G2 having the amino acid sequence of SEQ ID NO: 12

AFR-1D9 with the amino acid sequence of SEQ ID NO: 13

Example 4: Preparation of the antibody according to the invention:

Gene synthesis and obtaining constructs enabling the production of variable antibody fragments from llamas in a bacterial expression system.

Amino acid sequences of selected, most promising VHH fragments specific for the FOLR1 receptor were used to create constructs allowing for efficient expression in E. coli bacteria and enabling their purification on nickel resin.

Nucleotide sequences encoding the VHH genes from HIS-tag were synthesized by Eurofins. The genes encoding VHH have been optimized for efficient expression in bacteria. All genes were synthesized in two variants: with a signal sequence enabling protein expression and its efficient export to the periplasmic space and without a signal sequence allowing protein expression in the cytoplasm. The obtained genes were digested with the restriction enzymes NdeI and XhoI and then ligated into the pET30a expression vector prepared in the same way. The correctness of the final constructs was confirmed using Sanger sequencing.

Optimization of VHH production in E. coli

The obtained plasmids were transformed by chemical transformation into various previously prepared E. coli strains in order to optimize the production of VHH variants. The following strains were tested:

Table 2

E. coli strain Genotype E. coli BL21-CodonPhis(DE3)-RTL E. coli BF ^_ ompThsdS(r^~m^~} dcm Tct ^r gal D(DE3) endA Htc \argU ileY leuW Cam ^r ] SHuffle® T7 Exprcss lysY MiniF lysY (Cam ^R ) / ftniA2 lacZ::T7 genel [lon] ompT ahpC gal Zatt: :pNEB3-rl -cDshC (Spec ^R , lacF) AtrxB sulAl! Rfmcr73: :mimTnlQ—Yet ³ }2 [dcm] R(zgb-210::Tnl0 --Tet ^s ) cndAi Agor A(mcrC-mrr)H4: :IS10 Single Step (KRX) [F', traD36, ΔοιηρΡ, proA+B+, laclq, A(lacZ)M15] ΔοιηρΤ, endAl, recAl, gyrA96 (Nalr), thi-1, hsdR17 (rk-, mk+), e!4- (McrA- ), relAl, SLipE44, A(lac-proAB), A(rhaBAD)::T7 RNA polymerase.

ml of TB medium supplemented with kanamycin at a final concentration of 50 μg/ml was inoculated with an overnight bacterial culture in a ratio of 1:100 (culture from a single colony from transformation performed the previous day) and cultured until the optical density ODeoo = 0.6-0.8 at 37°C or 30°C, shaking 200 rpm. This is how the culture was prepared for each analyzed optimization condition. After achieving the expected culture density, the following induction conditions were tested:

a. Different strains of E. coli: E. coli BL21 -CodonPlus (DE3)-RIL, SHuffle® T7 Express lysY or Single

Step (KRX).

b. Different temperatures and induction times: 37°C or 30°C for 4 h and 25°C and 15°C overnight.

c. Different concentrations of IPTG inducer: 1 mM and 0.1 mM.

d. Different rhamnose concentration for Single Step (KRX) strain: 0.1% or 0.01%.

1 ml of sample was collected from each culture condition. The samples were centrifuged for 5 min at 10,000 x g at RT. The pellets were saved for analysis. The expression efficiency of the constructs was analyzed on an SDSPAGE gel under reducing and denaturing conditions.

Bacterial samples were prepared as follows:

The pellet from 1 ml of culture was suspended in 150 μΙ of BugBuster® Protein Extraction Reagent (Merck, cat. no. 70584). Incubated at RT (23°C) with shaking for 20 min. The samples were then centrifuged for 10 min, 10 g at 4°C to separate the soluble and insoluble fractions. 100 μΙ of the supernatant was transferred to a new tube and 12 μl of 6x concentrated sample buffer was added. The pellet was suspended in 150 μl of 1× concentrated sample buffer. Both fractions were incubated for 7 min at 95°C to denature the samples. The samples were then applied to a 4%/15% polyacrylamide gel. The gel was developed at 130 V for 20 min and then at 180 V until the gel was fully developed.

Large-scale VHH production

Optimization of the production of VHH particles allowed the selection of the best production conditions. The best selected variants were then produced in flasks and in the fermenter.

200 ml of Teriffic Broth medium supplemented with kanamycin at a final concentration of 50 μg/ml was inoculated with a single culture and cultured overnight at 37°C with shaking at 180 rpm. In the morning, 2 L of TB medium supplemented with kanamycin was inoculated with an overnight bacterial culture at a ratio of 1:100 and cultured to an optical density of OD600 = 0.6-0.8 at 37°C or 30°C with shaking at 200 rpm . The culture was then induced with 1 mM IPTG overnight at 25°C. Each protein was also produced in a 10 I fermenter. In this case, the culture was induced with 0.5 mM IPTG at 25°C. In each case, the correct protein was obtained in the soluble fraction.

Purification of VHH from E. Coli

The bacterial pellet containing overproduced VHH proteins with a polyhistidine tag was suspended in the buffer: TrisHCl pH 8.0, 250 mM NaCl, 10 mM imidazole, 5% glycerol. Cells were mechanically lysed using an overpressure of 20,000 psi. The cell lysate was then centrifuged at 4°C, 60 min, 14,000 rpm. Recombinant proteins were purified using affinity chromatography by applying the supernatant (soluble fraction) after centrifugation to a column containing a Ni-NTA His Bind bed, and then eluting the bound proteins with the same buffer containing a gradient increasing concentration of imidazole (10 - 500 mM). To specifically cleave the polyhistidine tag, purified protein fractions were treated with thrombin (1-3 U/mg) and incubated at 18-25°C. The cleaved tag was then removed by molecular filtration on a HiPrep Desalting column. This was also the step of replacing the buffer with the immunoconjugation formulation (200 mM NaCl, 100 mM sodium phosphate, pH 6.5). The preparations were then concentrated on an Amicon Ultra-15 Centrifugal Filter Unit with a 3 kDa membrane (Merck) to the concentration required for conjugation with the cytostatic.

Gene synthesis and preparation of a recombinant antibody binding to FOLR1 according to the seventh aspect of the invention

VHH molecules, tested for specificity of binding to receptors and safety of use, were used to obtain an antibody that binds to the FOLR1 receptor. Genes and ready-made constructs were obtained in an analogous manner as in the case of VHH fragments, using SEQ ID NO:21 of the first VHH-IgG chain and SEQ ID NO:22 of the second VHHIgG chain. The synthesized genes were cloned using the HindII and XhoI restriction enzymes into the pCDNA3.1+ expression vector. The addition of a signal sequence from human interleukin 2 allowed the protein to be exported to the medium, thus increasing expression and easy purification.

Two sets of mutations were introduced in the Fc fragments (according to Merchant et al.): S354C, T366W within one chain and Y349C, T366S, L368A, Y407V within the second chain. Their purpose is to facilitate the formation and folding of heterodimer forms during expression.

Co-transfections of the obtained plasmids into HEK293 cells were achieved using the Expifektamine reagent by mixing the diluted reagent into the mixture of two plasmids in the proportions suggested by the manufacturer.

Purification of Fc antibodies

The post-culture medium containing the isolated antibodies was injected onto a column containing a bed of protein A that specifically binds Fc fragments of antibodies (MabSelect Sure). Chromatographic separation was performed at a flow of 1 ml/min in PBS buffer pH 7.4. Antibodies bound to protein A were eluted with 100 mM sodium citrate buffer pH 3.3. The low pH also served to neutralize any viruses. Then the preparation was quickly neutralized by adding 10%

PL 244962 Β1 buffer 1.5 M TrisHCl pH 9.0. Finally, antibodies were formulated into PBS buffer pH 7.4 with 5% glycerol by molecular filtration on a column as above.

Example 5: Determination of VHH and antibody affinity

Binding of the obtained antibodies and VHH chains to the FOLR1 receptor using SPR - analysis of the specificity of interactions

The specific interactions of the obtained VHH molecules, antibodies containing them and immunoconjugates were determined using Surface Plasmon Resonance (SPR). The method involves immobilizing one of the interactors on the sensor surface, then various concentrations of solutions of the other interactor are passed through the space (chamber) above the sensor. The optical detection system is able to continuously record the increase in mass on the sensor with high sensitivity, thanks to which it is possible to track the kinetics of association and dissociation of interacting molecules in real time.

In this case, the target receptor FOLR1 was immobilized on the sensors. The attachment took place on the surface of one or two sensor chambers by covalent bonding of amine groups to carboxyl groups (amine coupling reactiori). Conjugation to the sensor surface was carried out until the desired occupancy level was obtained, which is calculated based on the ratio of molar masses and stoichiometry of both interactors. Two of the four sensor chambers were not occupied with a receptor and constituted a reference surface used to cut off non-specific signals. Sensors with immobilized receptors were used universally to determine all VHH clones and antibodies for a given target and as negative references for VHH clones and antibodies directed against other molecular targets. Impact measurements were carried out in two stages. In the first step, two concentrations (10 and 100 nM) of each VHH or antibody were injected onto the molecular targets. This was intended to demonstrate the dependence of the signal obtained on the device (RU level) on the concentration of the tested molecule. If the signals obtained were proportional to differences in concentrations, the range of concentrations used in the second stage to determine the kinetic parameters of interactions was determined based on their level. The typical concentration range used in the kinetic study included five samples of each VHH or antibody injected onto the immobilized receptor in a concentration range of 1-100 nM. The exception were some clones which, due to strong binding, required testing the concentration range of 0.25 - 25 nM to accurately calculate the parameters. During injection and analysis, the flow in the chamber was 30 μΙ/min. A single injection lasted 60 s and the subsequent dissociation lasted 600 s. To shorten the test time, the single cycle method was used. The obtained sensograms illustrating the association and dissociation of VHH and antibodies for molecular purposes in real time were used to calculate the interaction kinetics parameters using the 1:1 binding model. These included the association kinetics constant (ka or kon) - defining the specificity and binding strength, and the dissociation kinetics constant (kd or kof) defining the stability of the resulting complex. On their basis, the dissociation constant KD was calculated, which, expressed in moles, is a determinant of the bond strength compiling the remaining constants (kd/ka). The obtained values were used to create a ranking of the best molecules. Moreover, the lack of reaction with other receptors was an indicator of specificity.

Table 3. Kinetic parameters of the interaction of VHH clones and the antibody of the seventh aspect of the invention with their molecular target

VHH/ka antibody (I/Ms) kd (1/s) KD (nM) VHH 1G2 as shown in SEQ ID NO:12 9.25 x W ⁵ 2.07 x ^10-4 0.22 VHH 1D9 as shown in SEQ ID NO:13 1.49x10 ⁶ 1.13 x ^10-2 7.57 The antibody of the seventh aspect of the invention (Example 4) 2.48x10 ⁶ 2.31 x ^10-3 0.93

Example 6: Antibody immunoconjugation and immunoconjugate formulation

For the antibody according to the seventh aspect of the invention (Example 4), the immunoconjugation process with the Monomethylauristatin E (MMAE) molecule was optimized and performed. The selected immunoconjugation method is based on the reaction of an aldehyde obtained by oxidation of the N-terminal threonine with a hydrazide group belonging to the linker chain. The reaction steps are as follows: oxidation of N-terminal threonine (buffer: 200 mM NaCl, 100 mM sodium phosphate pH 6.5, 10 15-fold molar excess of NalCM,

PL 244962 Β1 minutes, RT, dark), protein reformulation (acetate buffer: 200 mM NaCl, 100 mM sodium acetate, pH 5.0), reaction with the 8310-MMAE substrate (other substrates were also tested) in the presence of aniline as a catalyst ( 30-fold molar excess of aniline, 5-fold molar excess of substrate, 40-42 h, RT, dark). This method has been optimized in terms of the amount of compound and catalyst as well as the reaction time. Preparations of individual proteins were obtained with yields of 68-83% in quantities sufficient to perform in vitro and in vivo experiments. The final formulation was neutral PBS pH 7.4 for administration to animals. Mass spectrometry (LC-ESI-MS) was used to confirm the mass of the obtained molecules.

In order to obtain a biological molecule that could carry a higher charge of a cytostatic compound, an immunoconjugation method was used, based on the reaction of reduced cysteines with maleimides contained in the linker. The protein was reduced (incubation with a 10-fold molar excess of TCEP, 18 h, RT) and then reacted with the MC Val Cit PAB MMAE compound (10-fold molar excess, incubation 5 h, RT). As a result of the modification, an average of 6 MMAE molecules were attached to each molecule of the tested antibody according to the invention. In order to increase the stability of the immunoconjugate, several formulations were tested, of which the final one was an acetate buffer (20 mM sodium acetate, pH 5.2, 5% (w/v) sucrose, 0.01% polysorbate 20), for which constant stability was observed in accelerated aging conditions. Mass spectrometry (LC-ESI-MS) was used to confirm the mass of the obtained molecule. The difference in mass resulting from the attachment of the cytostatic compound was also observed by SDS-PAGE electrophoresis.

Example 7: In vitro analysis of immunoconjugate and heterodimeric antibody

Analysis of the cytotoxic activity of the immunoconjugate

In order to determine the cytotoxic activity of the immunoconjugate obtained according to Example 6 and the antibody according to Example 4 in vitro against cells of selected cancer lines, an MTT viability test was performed. Human tumor-derived cell lines were obtained from ATCC and cultured in media recommended by the supplier. The breeding was carried out in standard conditions: 37°C, 5% CO2, 95% humidity. Cells were detached from the culture bottle using trypsin, complete medium was added and counted. The cell suspension was then seeded in a volume of 150 μΙ into 96-well plates in the amounts specified in Table 4 below.

Table 4

Name Type of cancer Medium Number per hole NIH:OVCAR-3 ATCC# HTB161 human ovarian cancer RPMl 1640+ 20% FBS + sodium pyruvate [ImM] + insulin [0.01 mg/ml] penicillin + streptomycin 6000 SK-OY-3 ATCC# HTB-77 human ovarian cancer McCoy's 5A + 10% FBS + penicillin + streptomycin 2000 SW620 ATCC# CCL-227 human colon cancer DMEM + 10% FBS. penicillin + streptomycin 6000 A549 ATCC# HTB185 human lung cancer RPMI 1640 + 10% FBS + 2mM sodium pyruvate + 1% solution of antibiotics penicillin and streptomycin A549 ATCC# HTB185

The plates were incubated for 24 hours at 37°C, 5% CO2, 95% humidity. The next day, the tested immunoconjugate according to Example 6 was added. For this purpose, a series of 10 dilutions were prepared for each preparation: immunoconjugate, antibody according to Example 4 (heterodimer of SEQ ID NO: 16 and SEQ ID NO: 17 chains), a mixture of the mentioned antibody + linker compound and relationship with the linker. The commercially available Trastuzumab-emtansine immunoconjugate (KADCYLA) was used as a control. Dilutions of the preparations were prepared in a mixture of complete media: RMPI-1640, McCoy's 5A and DMEM in a 1:1:1 ratio. Then, 50 μΙ of each preparation dilution was added per plate in triplicate. Control - 100% viability, consisted of cells in complete medium. The prepared plates were incubated for 72 hours at 37°C, 5% CO2, 95% humidity.

PL 244962 Β1

After this time, cell viability was assessed in vitro. For this purpose, a colorimetric MTT test was performed, which is used to measure cell proliferation, cell viability and cytotoxicity of all types of small-molecule compounds and biological molecules. The MTT test is based on the decomposition of the yellow tetrazolium salt MTT (3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide) into the water-insoluble purple formazan dye by succinyl-tetrazole reductase present in mitochondria. MTT reduction only occurs in living cells. The obtained results are presented in the form of IC50 values (the half maximal inhibitory concentration), i.e. the concentration of the tested preparation that reduces cell viability by half compared to the viability of control cells (constituting 100% viability). The MTT test protocol was as follows:

1. 20 μΙ of MTT solution [5 mg/ml] was added to each well.

2. The plates were incubated for 3 hours at 37°C, 5% CO2, 95% humidity.

3. The MTT medium was removed and 100 μΙ DMSO was added to dissolve the formazan crystals. A cell-free well with DMSO was used as a reference.

4. The plates were shaken until the formazan was completely dissolved.

5. The absorbance was measured at a wavelength of 570 nm (reference 690 nm).

6. The results were processed using GraphPad Prism 8 to determine the IC50 value.

The MTT viability assay was also used to determine in vitro degradation of the immunoconjugates of Example 6. The first step was to prepare 96-well plates with cells (all conditions identical to above). The cells were then treated with the immunoconjugate of Example 6, the antibody of Example 4 and the linker compound. 7 concentration points of each preparation were used. At appropriate time points, the medium above the cells was gently drained, the cells were rinsed with complete medium, then fresh medium was added and further incubation was carried out for up to 72 hours at 37°C, 5% CO2, 95% humidity.

The last stage was to perform the MTT test and determine the IC50 using the GraphPad Prism 8 program.

Name IC50 ng/ml, average values Immunoconjugate according to Example 6 Antibody according to Example 4 MMAE+linker control NIH:0VCAR-3 ATCC#HTB-161 0.15 3.24 2.56 0.24 SK-OV-3 ATCC# HTB-77 0.35 2.78 3.16 1.13 SW620 ATCC# CCL-227 0.83 3.15 2.18 1.16 A549 ATCC# HTB-185 >2000 >2000 5.16 >2000

Analysis of the attachment of the dimeric antibody presented according to Example 4 on the surface of human OVCAR-3 cancer cells performed by flow cytometry.

It was analyzed whether the obtained antibody, directed against the folic acid receptor (FOLR1) and labeled with the fluorochrome AF647, attached to OVCAR-3 cancer cells. The analysis was performed using flow cytometry. The experiment used 4 human cancer cell lines. The OVCAR-3 line and the SW620 line, which express high FOLR1 protein, were used as positive controls. The negative control consisted of 2 lines: A549 and A431, i.e. cells that do not express the FOLR1 protein.

PL 244962 Β1

Table 5

Name Type of cancer Medium NIH:OVCAR-3 ATCC# HTB-161 human ovarian cancer RPMI 1640+ 20% FBS + lmM sodium pyruvate + insulin [0.01 mg/ml] + 1% antibiotic solution penicillin and streptomycin SW620 ATCC# CCL-227 human colon cancer DMEM + 10% FBS + 1% penicillin and streptomycin antibiotic solution A549 ATCC# HTB-185 human lung cancer 0BR./MIN.I 1640 + 10% FBS + 2mM sodium pyruvate + 1% solution of antibiotics penicillin and streptomycin A431 ATCC# HTB-148 human skin cancer DMEM + 10% FBS + 1% penicillin and streptomycin antibiotic solution

Cell culture was performed on dishes with a diameter of 15 cm. The cells were detached using 0.5 mM EDTA solution. 1.5 * 10 ⁶ cells were taken for analysis. The cell suspension was centrifuged (5 min, 350 g, RT) and washed in phosphate-buffered saline (PBS) containing 3% fetal bovine serum (3% FBS). To block non-specific antibody binding sites, cells were incubated (10 min, 25°C) in a PBS solution with 3% FBS containing FC BŁOCK (2.5 pg/1*10 ⁶ cells/100 pL; BD Pharmingen # 564220). Cells were separated into separate tubes (2.5 * 10 ⁵ cells/l sample) and labeled for 30 minutes (25°C):

A) a commercially available antibody directed against the FOLR1 protein and labeled with the fluorochrome AF647 (1 pL/2.5 * 10 ⁵ cells; Novus Biologicals FAB5646R-100 μg);

B) 6 mM fluorochrome AF647 (1:10; 5 pL in 50 pL/ 2.5 * 10 ⁵ cells);

C) 43.6 pM of the antibody of Example 4 labeled with the fluorochrome AF647 (1:10; 10 pL in 100 pL/ 2.5 * 10 ⁵ cells);

D) 43.6 μΜ of the immunoconjugate according to Example 6 labeled with fluorochrome AF647 (1:20; 5 pL in 100 pL/ 2.5 * 10 ⁵ cells).

The control consisted of unlabeled cells. All samples were washed in 1 ml of PBS with 3% FBS three times. Cells were suspended in 0.5 ml of PBS with 3% FBS and analyzed on a FACS Canto flow cytometer. The experiment was performed in four biological replicates. The obtained data were analyzed using FlowJo software.

The analysis showed that the antibodies of Example 4 and the immunoconjugate of Example 6 directed against FOLR1 attach to cells that express high FOLR1 (OVCAR-3 and SW620 lines).

In vivo safety of the immunoconjugate antibody in a murine model

Determination of the maximum tolerated dose (MTD) in a mouse model.

Verification of the safety of the antibody according to Example 4 and the immunoconjugate according to Example 6 in vivo in a mouse model was carried out on the Hsd:Athymic Nude-Foxn1 ^nu strain, characterized by the lack of formation of cytotoxic effector cells and a deficiency of T lymphocytes. To determine the safety of the immunoconjugates used, an MTD study was performed (with Maximal Tolerated Dose), which involves selecting the maximum dose that ensures the most effective action of the tested molecule without causing severe side effects, including over 20% weight loss compared to the initial body weight of the mouse (all side effects are considered mild, moderate and severe were characterized by the Federation of European Laboratory Animal Science Associations - FelLASA).

The MTD study was performed according to a regimen known in the art with dose reduction or escalation depending on observed side effects.

MTD study

The MTD study was performed for a dose of 20 mg/kg of the immunoconjugate according to Example 6 and 20 mg/kg of the antibody according to Example 4 along with the following controls: positive 8310-MMAE (cytostatic drug monomethylauristatin E conjugated with a linker) and negative PBS + 2% DMSO ( the addition of 2% DMSO made it possible to exclude the influence of DMSO itself on the test results) in the qldx7 administration scheme (once a day, 7 days in a row) intravenously (into the tail vein). Since in the first phase (n=1) no severe side effects were observed with any of the tested antibodies and immunoconjugates, the study was performed on two more mice, so that a total of n=3. Body weight loss not exceeding 20% of the initial body weight, no other visible side effects such as: the lack of induced and spontaneous activity as well as the results of biochemistry and blood count made it possible to determine the maximum tolerated dose of approximately 20 mg/kg b.w. for the immunoconjugate according to Example 6 and the antibody according to Example 4.

For the immunoconjugate according to Example 6 and the antibody according to Example 4, a dose of 5 mg/kg b.w. was tested. In both cases, the tested mice showed negligible side effects after administration of the tested compounds.

Example 8: Determination of the anticancer effectiveness of tested compounds on human xenotransplants in a mouse model

This study involved inoculating human ovarian adenocarcinoma cells (OVCAR-3) suspended in a tumor-promoting medium into the right flank of Hsd:Athymic Nude - Foxn1 ^nu mice, measuring tumor growth, and then administering the immunoconjugate according to Example 6 and the antibody according to Example 4 to the subjects. tumor shrinkage.

The duration of the experiment depends on the growth rate of the tumors, because only when the average tumor volume V = 200 ^mm3 is reached, the mice are randomized and experimental groups are created so that each group has similar tumor volumes.

The immunoconjugate according to Example 6 and the antibody according to Example 4 were tested along with appropriate controls such as:

a) PBS buffer - as a negative control,

b) 8310-MMAE - as a positive control.

10 experimental groups were created, in which n = 5. The compounds were administered in a q2dx7 schedule (every other day, 7 times), a dose of 15 mg/kg b.w. was tested. The compounds were administered intravenously (tail vein).

The tumor size was measured using an electronic caliper, the tumor volume was calculated according to the formula: (a2 x b)/2, where a = shorter tumor diagonal (mm) and b = longer tumor diagonal (mm). Tumor growth inhibition was calculated using the formula:

TGI (%) (tumor growth inhibition) = (WT/WC) x 100 - 100%, where WT refers to the average tumor volume in the treated group, WC corresponds to the average tumor volume in the control group.

Experimental results are presented as mean (mean) ± SD (SD). All calculations and charts were prepared using the GraphPad Prism 5.0 program.

For the immunoconjugate, on day 50 after tumor implantation, a reduction in tumor volume was observed with an average TGI value of 95% +/- 5%, and for the antibody, a reduction in tumor volume with an average TGI value of 80% +/- 5% was observed. The positive control resulted in >98% TGI. No reduction in tumor volume was observed for the negative control.

PL 244962 Β1

Sequence list

SEQUENCE LISTING <110> Adamed Pharma S.A.

<120> Anti-FOLRl antibody and its use in medicine <130> BIOCONJNNOMED II <160> 24 <170> Patentln version 3.5 <210> 1 <211> 7 <212> PRT <213> Lama glama <400> 1

How much Thr Phe Ser Thr Thr Val 1 5 <210> 2 <211> 11 <212> PRT <213> Lama glama <400> 2

How much Ser Arg Val Gly Ser Thr Asn Tyr Val 1 5 10 <210> 3 <211> 5 <212> PRT <213> Lama glama <400> 3

Gly Asp Tyr Val Tyr 1 5 <210> 4 <211> 7 <212> PRT <213> Lama glama <400> 4

Ser Val Val Ser Asp Ala Val 1 5 <210> 5 <211> 11 <212> PRT <213> Lama glama

PL 244962 Β1 <400> 5

Val Ile Asn Thr Val Gly Ser Thr Ala Tyr Ala

5 10 <210>

<211>

<212>

<213>

PRT

Glama llama <400> 6

His Arg Ser Gly Tyr How much Tyr 1 5 <210> 7 <211> 26 <212> PRT <213> Lama glama <400> 7

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Ala Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

25 <210> 8 <211> 16 <212> PRT <213> Lama glama <400> 8

Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Glu Leu Vai Ala 15 10 15 <210> 9 <211> 38 <212> PRT <213> Lama glama <400> 9

Asp Ser Val Lys Gly Arg Phe Thr How much Ser Arg Asp Asn Ala Lys Asn 15 10 15

Thr Val Tyr Leu Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val

25 30

Tyr Tyr Cys Asn Ala Asn

PL 244962 Β1 <210> 10 <211> 38 <212> PRT <213> Lama glama <400> 10

Asp Ser Val Lys Gly Arg Phe Thr How much Ser Arg Asp How much Ala Lys Ser 15 10 15

Thr Ile Phe Leu Glu Thr Asn Ser Leu Lys Pro Glu Asp Thr Ala Val

25 30

Tyr Tyr Cys Asn Ala Asn <210> 11 <211> 11 <212> PRT <213> Lama glama <400> 11

Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser

5 10 <210> 12 <2U> 114 <212> PRT <213> Lama glama <400> 12

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Ala Gly Gly 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Thr Thr 20 2530

Val Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Glu Leu Val

4045

Ala How much Ser Arg Val Gly Ser Thr Asn Tyr Val Asp Ser Val Lys 50 5560

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 7580

Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 9095

PL 244962 Β1

Ala Asn Gly Asp Tyr Val Tyr Trp Gly Gin Gly Thr Gin Val Thr Val

100 105 110

Cheese Ser <210> 13 <211> 116 <212> PRT <213> Lama glama <400> 13

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Ala Gly Gly 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val Val Ser Asp Ala

2530

Val Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Glu Leu Val

4045

Ala Val How much Asn Thr Val Gly Ser Thr Ala Tyr Ala Asp Ser Val Lys 50 5560

Gly Arg Phe Thr How much Ser Arg Asp How much Ala Lys Ser Thr How much Phe Leu 65 70 7580

Glu Thr Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 9095

Ala Asn His Arg Ser Gly Tyr How Much Tyr Trp Gly Gin Gly Thr Gin Val

100 105110

Thr Val Ser Ser

115 <210> 14 <211> 232 <212> PRT <213> Artificial Seguence <220>

<223> Fc human IgG with heterodimerization mutations <400> 14

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 15 10 15

PL 244962 Β1

Pro Glu Leu Leu Gly Giy Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 2530

Lys Asp Thr Leu Met How Much Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 4045

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 5560

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin 65 70 7580

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin 85 9095

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105110

Leu Pro Ala Pro How Much Glu Lys Thr How Much Ser Lys Ala Lys Gly Gin Pro

115 120125

Arg Glu Pro Gin Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135140

Lys Asn Gin Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser 145 150 155160

Asp How Much Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr

165 170175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val

180 185190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe

195 200205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys

210 215220

Ser Leu Ser Leu Ser Pro Gly Lys

225230 <210> 15 <211> 232 <212> PRT <213> Artificial Seguence

PL 244962 Β1 <220>

<223> Fc human IgG with heterodimerization mutations <400> 15

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 15 1015

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 2530

Lys Asp Thr Leu Met How Much Ser Arg Thr Pro Glu Val Thr Cys Val Val

4045

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 5560

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin 65 70 7580

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin 85 9095

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105110

Leu Pro Ala Pro How Much Glu Lys Thr How Much Ser Lys Ala Lys Gly Gin Pro

115 120125

Arg Glu Pro Gin VaI Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

130 135140

Lys Asn Gin Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155160

Asp How Much Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr

165 170175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe

195 200205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys

210 215220

PL 244962 Β1

Ser Leu Ser Leu Ser Pro Gly Lys

225 230 <210> 16 <211> 356 <212> PRT <213> Artificial Seguence <220>

<223> first-chain heterodimer (VHHl+FcIgG) <400>

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Ala Gly Gly 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Thr Thr 20 2530

Val Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Glu Leu Val 35 4045

Ala How much Ser Arg Val Gly Ser Thr Asn Tyr Val Asp Ser Val Lys 50 5560

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 7580

Gin Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 9095

Ala Asn Gly Asp Tyr Val Tyr Trp Gly Gin Gly Thr Gin Val Thr Val 100 105110

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser

115 120125

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

130 135140

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 145 150 155160

Met How Much Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

165 170175

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

180 185190

PL 244962 Β1

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr

195 200205

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn

210 215220

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 225 230 235240

How much Glu Lys Thr How much Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin

245 250255

Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val

260 265270

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

275 280285

Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro

290 295300

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr 305 310 315320

Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val

325 330335

Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu

340 345350

Pro Gly Lys cheese

355 <210> 17 <211> 358 <212> PRT <213> Artificial Seguence <220>

<223> second heterodimer chain (VHH1+Fc IgG) <400> 17

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Ala Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val Val Ser Asp Ala

PL 244962 Β1

2530

Val Met Gly Trp Tyr Arg Gin Ala Pro Gly Lys Gin Arg Glu Leu Val

4045

Ala Val How much Asn Thr Val Gly Ser Thr Ala Tyr Ala Asp Ser Val Lys 50 5560

Gly Arg Phe Thr How much Ser Arg Asp How much Ala Lys Ser Thr How much Phe Leu 65 70 7580

Glu Thr Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 9095

Ala Asn His Arg Ser Gly Tyr How Much Tyr Trp Gly Gin Gly Thr Gin Val

100 105110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro

115 120125

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

130 135140

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

145 150 155160

Thr Leu Met How Much Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

165 170175

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

180 185190

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn

195 200205

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp

210 215220

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 225 230 235240

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu

245 250255

Pro Gin Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn

260 265270

PL 244962 Β1

Gin Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp How Much

275 280285

Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr

290 295300

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 305 310 315320

Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys

325 330335

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu

340 345350

Ser Leu Ser Pro Gly Lys

355 <210> 18 <211> 485 <212> PRT <213> Artificial Sequence <220>

< 223> FOLR1 mature chain fused with the Fc fragment and IgG signal sequence < 400> 18

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1015

Val His Ser Arg How Much Ala Trp Ala Arg Thr Glu Leu Leu Asn Val Cys 20 2530

Met Asn Ala Lys His His Lys Glu Lys Pro Gly Pro Glu Asp Lys Leu

4045

His Glu Gin Cys Arg Pro Trp Arg Lys Asn Ala Cys Cys Ser Thr Asn 50 5560

Thr Ser Gin Glu Ala His Lys Asp Val Ser Tyr Leu Tyr Arg Phe Asn 65 70 7580

Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys Lys Arg His Phe Ile 85 9095

PL 244962 Β1

Gin Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly Pro Trp How Much

100 105110

Gin Gin Val Asp Gin Ser Trp Arg Lys Glu Arg Val Leu Asn Val Pro

115 120125

Leu Cys Lys Glu Asp Cys Glu Gin Trp Trp Glu Asp Cys Arg Thr Ser

130 135140

Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp Thr Ser Gly 145 150 155160

Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gin Pro Phe His Phe Tyr

165 170175

Phe Pro Thr Pro Thr Val Leu Cys Asn Glu How Much Trp Thr His Ser Tyr

180 185190

Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys How Much Gin Met Trp

195 200205

Phe Asp Pro Ala Gin Gly Asn Pro Asn Glu Glu Val Ala Arg Phe Tyr

210 215220

Ala Ala Ala Met Glu Asn Leu Tyr Phe Gin Ser Gly Gly Ser Gly Ser 225 230 235240

Gly Gly Gly Glu Pro Lys Ser Gin Asp Lys Thr His Thr Cys Pro Pro

245 250255

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

260 265270

Pro Lys Pro Lys Asp Thr Leu Met How Much Ser Arg Thr Pro Glu Val Thr

275 280285

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

290 295300

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315320

Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

325 330335

Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

PL 244962 Β1

340 345350

Asn Lys Ala Leu Pro Ala Pro How Much Glu Lys Thr How Much Ser Lys Ala Lys

355 360365

Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp

370 375380

Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395400

Tyr Pro Ser Asp How Much Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu

405 410415

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

420 425430

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly

435 440445

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

450 455460

Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Gly Ser Gly Gly Gly His

465 470 475480

His His His His His

485 <210> 19 <211> 696 <212> DNA <213> Artificial Sequence <220>

<223> The first FC chain IGG <400> 19 GAACCGAAA GCTGCGATAA AACCATACC TGCCCGT GAAG TGACCTGCGT GGTGTGTGAT GTGAGCATG AAGATCCGGA AGTGAATTT 180 AACTGTATG TGGATGCGT GGAAGTGCAT CATCAGGA TTGGCTGAAC 300 GGCAAAGAAT ATAATGCAA AGTGAGCAAC AAAGCGCTGC cggcgccgat tgaaaaaacc 360 attagcaaag cgaaaggcca gccgcgcgaa ccgcaggtgt gcaccctgcc gccgagccgc 420

PL 244962 Β1 gatgaactga ccaaaaacca ggtgagcctg agctgcgcgg tgaaaggctt ttatccgagc 480 gatattgcgg tggaatggga aagcaacggc cagccggaaa acaactataa aaccaccccg 540 ccggtgctgg atagcgatgg cagctttttt ctggtgag ca aactgaccgt ggataaaagc 600 cgctggcagc agggcaacgt gtttagctgc agcgtgatgc atgaagcgct gcataaccat 660 tatacccaga aaagcctgag cctgagcccg ggcaaa 696 <210> 20 <211> 695 <212> DNA <213 > Artificial Sequence <220>

<223> Second FC chain IGG <400> 20 GAACCAAA GCTGCGATAA AACCATACC TGCCCCGT GCCCGCCC GGAACTGCTG 60 GGCGCCCGA GCGTGTTTC GTTTTCCCG AAACCGAAAG ATACCCG GAAG TGACCTGCGT GGTGTGTGAT GTGAGCATG AAGATCCGGA AGTGAATTT 180 AACTGTATG TGGATGCGT GGAAGTGCAT CATCAGGA TTGGCTGAAC 300 GGCAAAGAAT ATAATGCAA AGTGAGCAAC AAAGCGCTGC CGGCCCGAT TGAAAAAAC 360 Attagcaaag CGAAAGCCA GCCGCGCAA CCGCAGTGTTGT ATACCCTGCC GCCCGCCGC 420 GaATGGGA AAGCAACGC CAGCCGGAA ACACTATAA AACACCCG 540 CCGGTGCTGG ATAGCGATGGGGGGG CTGTTTTTTTTTT CTGTATAGCA AACTGACCGT GGATAAAGC 600 CGCTGCAD Accat 660 Tatacccaga Aaagcctgag CCTGAGCCG GGCAA 695 <210> 21 <211> 1068 <212> DNA <213> Artificial Sequence <220>

<223> heterodimer first chain (VHH1+Fc IgG) <400> 21 gaagtgcagc tggtggaaag cggcggcggc ctggtgcagg cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggcat tacctttagc accaccgtga tgggctggta tcgccaggcg 120 ccggg caaac agcgcgaact ggtggcgatt attagccgcg tgggcagcac caactatgtg 180

Pl 244962 β1 Gatagcgtga AAGGCGCTT TACCATTAGC CGCGATAACG CGAAAAC CGTGTATCTG 240 Cagatgaaca GCCTGAAAC CCGTGAGCA GCGGCGGCGG CGGCAGCGC 360 GGCGGCGGCA GCGAACGAA AAGTGCGAT AAAACCATA CCTGCCCGC ATACCCTG 480 ATGATTAGC GCACCCGGA AGTGACCTGC GTGGTGTGGGT ATGTGCCA TGAAGATCCG 540 GAAGTGAAAT TTAACTGGTA TGTGGATGGC GTGGTGC ATAACGCGAA AACAAACG 600 CGCGAACAC AGTATAACAG CACCTATCGC GTGGTGAGCG TGCTGACGT GCTGCATCAG 660 GATTGGTGA ACGGCAAAG CCGCGCG AACCGCAGGT GTGCACCCTG 780 CCGCCGAGCC GCGATGAACT GACCAAAAAC accacccccccgtgct ggatagcgat ggcagcttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt GCCAGCGCG 1020 ctgcataacc attataccca gaaaagcctg agcctgagcc cgggcaaa 1068 <210> 22 <211> 1074 <212> DNA <213> Artificial Seguence <220>

<223> Second heterodimer chain (VHH1+FC IGG) <400> 22 GAAGTGCAG TGGTGGAAG CGGCGCGGC CTGGTGCAG TCGCCAGCG 120 CCGGGCAAAAC AGCGCGAACT GGTGCGGTG Attaaccg TGGGCACAC CGCGTATGCG 180 Gatagcgtga AAGGCCGCTT TACCATTAGC CGCGATTTG CGAAAGCAC Cattttctg 240 GAACAAC ACC GCGGTGTATT ATTGCAACGC GAACCATCGC 300 AGCGGCTATA TTTATTGGGGGGGGCACC CAGGTGACG TGAGCAGCGG CGGCGCGC 360 AGCGCGCG GCGCAD GGCCCGAGC GTGTTTTGT TTCCCGCAACH CGAAAAC 600 AAACCGCGCG AAGAACAGTA TAAACAGCACCCA DATCGCGTGG TGAGCGTGCT GACCGTGTG 660

PL 244962 Β1 catcaggatt ggctgaacgg caaagaatat aaatgcaaag tgagcaacaa agcgctgccg 720 gcgccgattg aaaaaaccat tagcaaagcg aaaggccagc cgcgcgaacc gcaggtgtat 780 accctgccgc cgtgccgcga tgaactgacc aaaaac cagg tgagcctgtg gtgcctggtg 840 aaaggctttt atccgagcga tattgcggtg gaatgggaaa gcaacggcca gccggaaaac 900 aactataaaa ccaccccgcc ggtgctggat agcgatggca gcttttttct gtatagcaaa 960 ctgaccgtgg ataaaagccg ctggcagcag ggcaacgtgt ttagctgcag cgtgatgcat 1020 gaagcgctgc ataaccatta tacccagaaa agcctgagcc tgagcccggg caaa 1074 <210> 23 <2U> 342 <212> DNA <213> Lama glama <400> 23 gaagtgcagc tggtggaaag cggcggcggc ctggtgcagg cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggcat tacctttagc accaccgtga ggta tcgccaggcg 120 ccgggcaaac agcgcgaact ggtggcgatt attagccgcg tgggcagcac caactatgtg 180 gatagcgtga aaggccgctt taccattagc cgcgataacg cgaaaaacac cgtgtatctg 240 cagatgaaca gcctgaaacc ggaagatacc gcggtgtatt attgcaacgc gaacggcgat 300 tatgtgtatt ggggccaggg cacccaggtg accgtgagca gc 342 <210> 24 <211> 348 <212> DNA <213> Lama glama <400> 24 gaagtgcagc vol ggtggaaag cggcggcggc ctggtgcagg cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggcag cgtggtgagc gatgcggtga tgggctggta tcgccaggcg 120 ccgggcaaac agcgcgaact ggtggcggtg attaacaccg tgggcagcac cgcgtatgcg 180 gatagcgtga aaggccgctt taccattagc cgcgatattg cgaaaagcac catttttctg 240 gaaaccaaca gcctgaaacc ggaagatacc gcggtgtatt attgcaacgc gaaccatcgc 30 0 agcggctata tttattgggg ccagggcacc caggtgaccg tgagcagc 348

Claims (14)

1. An anti-FOLR1 antibody comprising first and second antibody fragments, wherein - the first fragment of the anti-FOLRI antibody comprises a first VHH chain operably linked to the first Fc constant heavy chain fragment of a human IgG, and - the second anti-FOLRI antibody fragment comprises a second VHH chain operably linked to a second human IgG Fc constant heavy chain fragment, and wherein - said first VHH chain contains a complementarity determining region (CDR) consisting of CDR1 as shown in SEQ ID NO.: 1, CDR2 as shown in SEQ ID NO.: 2 and CDR3 as shown in SEQ ID NO.: 3 and a framework region ( FR) consisting of FR1 as set out in SEQ ID NO: 7, FR2 as set out in SEQ ID NO: 8, FR3 as set out in SEQ ID NO: 9 and FR4 as set out in SEQ ID NO: 11, and - said second VHH chain contains a complementarity determining region (CDR) consisting of CDR1 as shown in SEQ ID NO.: 4, CDR2 as shown in SEQ ID NO.: 5 and CDR3 as shown in SEQ ID NO.: 6 and a framework region ( FR) consisting of FR1 as set out in SEQ ID NO: 7, FR2 as set out in SEQ ID NO: 8, FR3 as set out in SEQ ID NO: 10 and FR4 as set out in SEQ ID NO: 11. 2. The antibody of claim 1. 1, wherein the first VHH chain has the sequence of SEQ ID NO.: 12 and the second VHH chain has the sequence of SEQ ID NO.: 13. 3. The anti-FOLR1 antibody of claim 1. 1 or 2, wherein the first human IgG Fc constant heavy chain fragment has a sequence as set forth in SEQ ID NO: 14 and the second human IgG constant Fc heavy chain fragment has a sequence as set forth in SEQ ID NO: 15. 4. The anti-FOLR1 antibody of claim 1. 1, 2 or 3, wherein the first anti-FOLR1 antibody fragment has a sequence as set forth in SEQ ID No: 16, the second anti-FOLR1 antibody fragment has a sequence as set forth in SEQ ID No: 17. 5. An isolated polynucleotide encoding a protein selected from the group consisting of: - a first fragment of the anti-FOLR1 antibody as set out in SEQ ID NO: 21; - a second anti-FOLR1 antibody fragment as set forth in SEQ ID NO: 22; - a first fragment of the constant Fc heavy chain of human IgG as shown in the amino acid sequence of SEQIDNO: 19; - a second human IgG Fc heavy chain constant fragment as shown in the amino acid sequence of SEQ ID NO: 20; - the first anti-FOLR1 VHH chain as shown in SEQ ID NO: 23; - a second anti-FOLR1 VHH chain as shown in SEQ ID NO: 24. 6. The isolated polynucleotide according to claim 1. 5, characterized in that said polynucleotide has the nucleotide sequence of SEQ ID NO: 21 and/or SEQ ID NO: 22. 7. An expression vector comprising a polynucleotide as defined in claim 1. 5 or 6. 8. A host cell, characterized in that said host cell comprises an expression vector as defined in claim 1. 7, or a polynucleotide as defined in claim 7. 5 or 6 integrated into the host cell genome. 9. A method of producing an anti-FOLR1 antibody comprising the steps of: (a) culturing a host cell as defined in claim 1. 8 under conditions suitable for producing the antibody, thereby obtaining a culture containing said anti-FOLR1 antibody; and (b) isolating or recovering said anti-FOLR1 antibody from said culture. 10. An immunoconjugate, characterized in that said immunoconjugate comprises: (a) an anti-FOLR1 antibody as defined in any one of claims. 1 to 4; and (b) a conjugated moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide and an enzyme. 11. An antibody as defined in claim 1. 1 to 4 or an immunoconjugate as defined in claim 1. 10 for use as (a) an agent for detecting a FOLR1 molecule; or (b) a drug for treating cancer. PL 244962 Β1 12. Pharmaceutical composition comprising: (i) an anti-FOLR1 antibody as defined in claim 1. 4, or an immunoconjugate as defined in claim 4. 10; and (ii) a pharmaceutically acceptable carrier. 13. The pharmaceutical composition according to claim 1. 12 for use in the treatment of cancer. 14. The use of an anti-FOLR1 antibody as defined in claim 1. 4, or an immunoconjugate as defined in claim 4. 10 for preparing a detection agent, plate or kit, wherein said detection agent, plate or kit is used to detect the FOLR1 protein in a test sample.