EP4229090A1 - Anti-gpc4 single domain antibodies - Google Patents

Abstract

The present disclosure relates to anti-GPC4 single domain antibodies (sdAb) and variants thereof, notably linked directly or not to a compound of interest, multivalent binding compounds or chimeric antigen receptor and to their use in therapy or as diagnostic and/or detection compounds.

Description

Anti-GPC4 single domain antibodies

FIELD OF THE INVENTION

The present disclosure relates to anti-GPC4 single domain antibodies (sdAb) and variants thereof, notably linked directly or not to a compound of interest and/or included in chimeric antigen receptor and to their use in therapy or as diagnostic and/or detection compounds.

DETAILED DESCRIPTION

Glypicans have arised as key modulators of signaling activities given their structural features (1,2,3). Glypicans, which in mammals account for a family of six different members, are composed of a core protein to which two long linear glycosaminoglycan heparan sulfate (HS) chains are covalently linked to the C-terminal portion of Glypicans (1, 2, 3). They are anchored to the cell surface by a glycosylphosphatidylinositol (GPI) lipid anchor and can be released into the extracellular space following cleavage by various enzymes (1, 2, 3). Previous studies suggest three main Glypican functions. First, they act as coreceptors for the Wnt, FGF, Hh and BMP pathways by promoting ligand receptor interaction (1, 4). Second, Glypicans alter the diffusion of signaling molecules in the extracellular space, thus contributing to generate morphogen gradients (1, 4, 5). Third, HSPGs can be cleaved or shed from the cell membrane, changing ligand concentration, availability to adjacent cells and its temporal action (1, 4, 6). In summary, Glypicans are important for signaling activation and to generate morphogen gradients by titrating temporal and spatial availability as well as the amplitude and duration of the signaling input. As consequence, functional studies involving loss- and gain- of Glypican functions have highlighted their key implications in developmental processes and human pathologies such as cancer (7, 8, 9, 10).

More recently, targeting Glypican activity has also emerged as a strategy to fine tune activation of signaling pathways in physiological conditions and to prevent pathological processes. For example, the Glypican family member, Glypican-3 (GPC3) has attracted attention as a novel therapeutic target for hepatocellular carcinoma (11) whereas the blockage of the member Glypican-2 (GPC2) is currently evaluated for the neuroblastoma treatment (12). The inventors have recently demonstrated that another Glypican, Glypican-4 (GPC4) is an attractive target to modulate stem cell (SC) functions as it down-regulation permits to impact on the processes of self-renewal and differentiation of SCs (13). In particular, they have demonstrated that pluripotent SCs (PSCs) such human induced PSCs (hiPSCs) and mouse embryonic SCs (mESCs) with reduced GPC4 protein levels acquire unique biological states that enables: 1) maintenance of self-renewal/pluripotency in sternness conditions, 2) efficient lineage entry in differentiation conditions (mesoderm, ectoderm, or endoderm), 3) loss of tumorigenicity in xenografts (14). They have also shown that down-regulation of GPC4 in PSCs can promote the application of hPSC in medicine. By using Parkinson’s disease as paradigm, they have notably demonstrated that PSCs with reduced GPC4 activity have higher in vitro differentiation propensity towards midbrain dopaminergic (mDA) neurons, which correspond to the neuronal cell type degenerating in PD patients (15). Moreover, when transplanted in the brain of a rat models for Parkinson’s disease (PD) these GPC4 mutant PSCs generate greater mDA neuron numbers capable to rescue the motor defects characterizing PD rat models (15). Importantly, these behavioral improvements occur without causing tumor side effects observed using control cells (15). Thus, the biological state conferred to PSCs by GPC4 down-regulation appears compatible with efficient and safe production of mDA neurons with potential clinical relevance for PD therapy (15).

Besides SCs, targeting GPC4 may also serve as a promising strategy for other human pathologies, notably for the treatment of cancer. For example, proteasome-dependent ubiquitination of GPC4 repress colorectal tumorigenesis by inhibiting beta-catenin/c-myc signaling (16) whereas loss-of GPC4 activity in pancreatic cancer cells sensitizes them to chemotherapeutic agents and attenuated stem cell-like properties (17).

High GPC4 serum levels are also associated with metabolic disorders such as high body fat content, insulin resistance and nonalcoholic fatty liver disease (18, 19).

Taken together these studies highlight GPC4 as a potential target for therapeutic strategies of different human disorders. They also reveal that GPC4 might become a promising diagnostic and prognostic marker for disease prediction, patient’s stratification, and/or evaluation of effective therapeutic protocols. Development of therapeutic and diagnostic tools targeting GPC4 is therefore highly clinically relevant.

Current clinical practices make use of antibodies for disease diagnosis and treatment (20). Besides the conventional heterotetrameric antibodies, the immune system of Camelids (e.g. llamas) contains heavy-chain-only antibodies that are devoid of light chains (21). The variable antigen-binding domains of these antibodies (VHH) display a full antigen-binding specificity. They have advantages over conventional antibodies in that they are much smaller (15 kDa versus 150-160 kDa) and can be isolated and produced separately as single domain antibodies (sdAb); 21). sdAbs have also low immunogenicity, a unique binding capability, and high solubility and stability. Because of their small size, sdAbs can penetrate small clefts and cavities (22, 23). Moreover, having a long flexible CDR3 loop, sdAbs can bind conformational epitopes that are often inaccessible to conventional types (24). This property increases their probability of being antigen-blocking reagents. Nbs targeting the GPC3 and GPC2 have efficiently identified by screening a combinational engineered human VH single domain phage display library (10, 25), however to the inventor’s knowledge single domain antibodies targeting GPC4 have never been described.

Thus, there remains an unmet need to improve and diversify current diagnostic and therapeutic tools targeting GPC4.

SUMMARY OF THE INVENTION

The present application now provides single domain antibodies specifically binding to GPC4 with a high affinity.

The inventors have now generated the first two GPC4-targeting single domain antibodies (sdAb) binding hGPC4 in vitro and/or in cells upon hGPC4cDNA transfection, with nanomolar affinities, named herein as RB. Following the generation of a bivalent form of RB, obtained upon its fusion to the Fc domain of the human IgG, they demonstrated that this sdAb- Fc form is a high-affinity sdAb specific for endogenously expressed hGPC4 and that recognizes a conformational epitope in the native hGPC4 protein. These GPC4-targeting sdAbs therefore not only represent promising tools to enhance the therapeutic potential of hPSCs without involving genetic manipulation but should also provide therapeutic benefit in different human disorders involving changes in GPC4 levels.

Thus, the present disclosure relates to a single domain antibody (sdAb) directed against GPC4, wherein said anti-GPC4 sdAb has the following formula FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4, and wherein the CDRs are selected from: a CDR1 of SEQ ID NO: 1; a CDR2 of SEQ ID NO:2 and a CDR3 of SEQ ID NOG, or a CDR1 of SEQ ID NO:4; a CDR2 of SEQ ID NOG and a CDR3 of SEQ ID NOG, In more particular embodiments, the present disclosure contemplates humanized anti-GPC4 having:

- a CDR1 of SEQ ID NO:1; a CDR2 of SEQ ID NO:2 and a CDR3 of SEQ ID NO:3;

- a CDR1 of SEQ ID NO:4; a CDR2 of SEQ ID NO:5 and a CDR3 of SEQ ID NO:6; a sequence of SEQ ID NO: 15; a sequence of SEQ ID NO: 16; a sequence of SEQ ID NO: 19; a sequence having at least 90 % identity with SEQ ID NO: 15 a sequence having at least 90 % identity with SEQ ID NO: 16; a sequence having at least 90 % identity with SEQ ID NO: 19;

- a CDR1 of SEQ ID NO: 1 ; a CDR2 of SEQ ID NO:2 and a CDR3 of SEQ ID NO:3 and further having one or more conservative amino acid modifications in one or more of these CDRs; or

- a CDR1 of SEQ ID NO:4; a CDR2 of SEQ ID NO:5 and a CDR3 of SEQ ID NO:6 and further having one or more conservative amino acid modifications in one or more of these CDRs.

The anti-GPC4 sdAb of the present disclosure can be linked directly or indirectly, covalently or non-covalently to a compound of interest selected from a nucleic acid, a polypeptide or a protein, a virus, a toxin and a chemical entity. In some embodiments, the anti-GPC4 sdAb is linked directly or indirectly, covalently or non-covalently to a diagnostic compound selected from an enzyme, a fluorophore, a NMR or MRI contrast agent, a radioisotope and a nanoparticle. In some embodiments, the anti-GPC4 sdAb is linked directly or indirectly, covalently or non-covalently to a compound selected from cytotoxic agents, chemotherapeutic agents, radioisotopes, targeted anti-cancer agents, immunotherapeutic agents (such as immunosuppressants or immune stimulators), and lytic peptides.

The GPC4 sdAb can also be fused to an immunoglobulin domain, in particular to an Fc domain.

The present disclosure also encompasses multivalent or multispecific binding compounds comprising at least a first sdAb consisting in a GPC4 sdAb as herein described, and further comprising another sdAb binding to the same or to a second antigen.

Optionally, the at least second sdAb is a GPC4 as herein described. Optionally, the first sdAb is located at the N-terminus of the second sdAb or wherein the first sdAb is located at the C-terminus of the second sdAb.

Thus, the present disclosure encompasses multivalent binding compounds wherein the first and at least second sdAbs are in a tandem format; optionally wherein the first and second sdAbs are constructed in a head to tail tandem format.

The present disclosure also encompasses multivalent binding compounds in a sdAb-Fc format; optionally wherein the multivalent binding compound is in a bivalent sdAb-Fc format; more particularly wherein, the bivalent sdAb-Fc compound comprises a first and a second sdAb which are identical and preferably which are sdAbs as herein disclosed.

The present disclosure further encompasses a chimeric antigen receptor (CAR) comprising (a) an antigen binding domain comprising at least a first sdAb consisting in the GPC4 sdAb as herein or a multivalent, or multispecific compound as herein described, (b) a transmembrane domain; and (c) an intracellular domain.

The present disclosure also encompasses an isolated nucleic acid comprising a nucleic acid sequence encoding a anti-GPC4 sdAb or a CAR as herein described, which is advantageously linked to a heterologous regulatory control sequence.

The present disclosure also encompasses vectors comprising the nucleic acids as herein disclosed, host cells comprising thereof, isolated cells or population of cells expressing an anti- GPC4 sdAb, or a CAR as herein disclosed. In some embodiments, said cells are immune cells than can be selected from macrophages, NK cells and T cells, notably CD4+/CD8+, TILs/tumor derived CD8 T cells, central memory CD8+ T cells, Treg, MAIT, and Yδ T cell.

The therapeutic product of the present disclosure, including an anti-GPC4 sdAb, notably a humanized anti-GPC4 sdAb, a multivalent binding compound, a CAR, a nucleic acid, a vector, a host cell, an isolated cell or cell population as defined herein can be used in therapy, notably in the treatment of a disease selected from a proliferative disease, a neurodegenerative disease or a metabolic disorder or for cell-based replacement therapy in a subject in need thereof. They can notably be used in cellular therapy of cancer and/or in combination with other therapy, notably another cancer therapy. Typically, the disease is a GPC4 associated disease, notably a GPC4 associated cancer such as a colorectal cancer or a pancreatic cancer.

The present disclosure also encompasses the use of an anti-GPC4 sdAb as defined in any one of claims 1-4 or of a multivalent binding compound as defined in any one of claims 5-6, in vitro or ex vivo, to promote self-renewal and differentiation of stem cells (SCs); optionally the stem cells (SCs) are embryonic stem cells or pluripotent stem cells, such as induced PSCs and notably human iPSCs; optionally said sdAb or multivalent binding compound promotes efficient lineage entry, notably in mesoderm, ectoderm, or endoderm lineage.

The present disclosure further pertains to a method of producing differentiated stem cells (SCs), notably midbrain dopaminergic neurons (mDA neurons), comprising culturing said SCs in the presence of an anti-GPC4 sdAb as defined in any one of claims 1-4 or of a multivalent binding compound as defined in any one of claims 5-6;

Optionally wherein said SCs are pluripotent stem cells (PSCs), notably human induced PSCs (hiPSCs)

The present disclosure also pertains to an anti-GPC4 sdAb, notably a humanized anti- GPC4 sdAb, a multivalent binding compound, a CAR, a nucleic acid, a vector, a host cell, an isolated cell or cell population as defined herein for use as a medicament.

The present disclosure further includes the use of anti-GPC4 sdAb as herein described for the detection or monitoring of GPC4 is a biological sample.

The present disclosure also encompasses:

An in vitro method for diagnosing a GPC4-associated disease in a subject, wherein the method comprises: contacting a biological sample obtained from the subject with an anti-GPC4 single domain antibody as defined herein; determining the level of expression of GPC4 in said sample by detecting the binding of said anti-GPC4 sdAb to GPC4 expressed by the sample; and comparing the level of expression of GPC4 in said sample with a reference value.

An in vitro method for determining the eligibility of a subject to a treatment with an anti-GPC4 therapy, wherein the method comprising: determining the presence or expression level of GPC4 in a sample obtained from the subject by contacting the sample with an anti-GCP4 sdAb as herein defined and detecting the presence of the bound sdAb;

In some embodiment, the anti-GPC4 therapy is an anti-GPC4 sdAb, a multivalent binding compound or a chimeric antigen receptor as defined herein.

An in vitro method for monitoring a treatment efficacy in a subject receiving a treatment for a GPC4-associated disease, wherein the method comprises determining in a biological sample of said subjectn at two or more time points, the level of expression of the GPC4 protein, and wherein the determination of level of expression of the GPC4 protein in a biological sample of the subject comprises: contacting a biological sample obtained from the subject with an anti-GPC4 single domain antibody as defined herein; determining the level of expression of GPC4 in said sample by detecting the binding of said anti-GPC4 sdAb to GPC4 expressed by the sample; and comparing the level of expression of GPC4 in said sample with a reference value. A method for identifying a subject suffering from a GPC4-associated disease, who is likely to respond to a treatment, the method including: determining the presence or expression level of GPC4 in a sample obtained from said subject by contacting the sample with an anti-GPC4 sdAb as defined herein, and detecting the presence of the bound sdAb, wherein the presence or expression level of GPC4 in said sample indicates that the subject is likely to respond to the treatment.

A method for predicting the responsiveness of an individual suffering from a cancer to a treatment with an anti-cancer therapy, notably an anti-GPC4 therapy, comprising: determining the presence or expression level of GPC4 in a sample obtained from said subject by contacting the sample with an anti-GPC4 sdAb, as defined herein; and detecting the presence of the bound sdAb wherein the presence or expression level of GPC4 in the sample indicates that the subject is more likely to respond to treatment with the anti-cancer therapy.

In some embodiment, the anti-cancer treatment includes an anti-GPC4 sdAb, a multivalent binding compound, or a chimeric antigen receptor as defined herein.

DETAILED DESCRIPTION

Definitions:

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be exhaustive. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. The term "comprising" as used herein is synonymous with "including" or "containing” and is inclusive or open-ended and does not exclude additional, uncited members, elements or method steps.

Unless specifically stated or obvious from context, as used herein, the term “about” is to be understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

As used herein, the term "isolated" refers to a substance or entity that has been (1 ) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components.

The "isolated" products of the present disclosure, including isolated nucleic acids, proteins, polypeptides, antibodies or cells are not products of nature (i.e., "non-naturally occurring"). Rather, the "isolated" nucleic acids, proteins, polypeptides, and antibodies of the present disclosure are "man-made" products. The "isolated" products of the present disclosure can be "markedly different" or "significantly different" from products of nature. By way of a non-limiting example, the isolated nucleic acids may be purified, recombinant, synthetic, labeled, and/or attached to a solid substrate. Such nucleic acids can be markedly different or significantly different than nucleic acids that occur in nature. By way of further non-limiting example, the "isolated" proteins, polypeptides, and antibodies of the present disclosure may be purified, recombinant, synthetic, labeled, and/or attached to a solid substrate. Such proteins, polypeptides, and antibodies can be markedly different or significantly different from proteins, polypeptides, and antibodies that occur in nature.

The term "polynucleotide", "nucleic acid molecule", "nucleic acid", or "nucleic acid sequence" refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native intemucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, doublestranded, triple-stranded, quadruplexed, partially double- stranded, branched, hairpinned, circular, or in a padlocked conformation. The nucleic acid (also referred to as polynucleotides) may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in "locked" nucleic acids.

A "synthetic" RNA, DNA or a mixed polymer is one created outside of a cell, for example one synthesized chemically.

The term "nucleic acid fragment" as used herein refers to a nucleic acid sequence that has a deletion, e.g., a 5'-terminal or 3'-terminal deletion compared to a full-length reference nucleotide sequence. In an embodiment, the nucleic acid fragment is a contiguous sequence in which the nucleotide sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. In some embodiments, fragments are at least 10, 15, 20, or 25 nucleotides long, or at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, or 150 nucleotides long. In some embodiments a fragment of a nucleic acid sequence is a fragment of an open reading frame sequence. In some embodiments such a fragment encodes a polypeptide fragment (as defined herein) of the protein encoded by the open reading frame nucleotide sequence. The nucleic acid can be purified. Preferably, the purified nucleic acid is more than 50%, 75%, 85%, 90%, 95%, 97%, 98%, or 99% pure. Within the context of the present disclosure, a purified nucleic acid that is at least 50% pure means a purified nucleic acid sample containing less than 50% other nucleic acids. For example, a sample of a plasmid can be at least 99% pure if it contains less than 1 % contaminating bacterial DNA.

The term "operably linked" in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof. Further, a polypeptide may comprise a number of different domains each of which having one or more distinct activities. For the avoidance of doubt, a "polypeptide" may be any length greater two amino acids.

The term "peptide" as used herein refers to a short polypeptide, e.g., one that typically contains less than about 50 amino acids and more typically less than about 30 amino acids. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As thus defined, "isolated" does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from a cell in which it was synthesized.

The protein or polypeptide can be purified. Preferably, the purified protein or polypeptide is more than 50%, 75%, 85%, 90%, 95%, 97%, 98%, or 99% pure. Within the context of the present disclosure, a purified protein that is more than 50% (etc.) pure means a purified protein sample containing less than 50% (etc.) other proteins. For example, a sample of a protein comprising can be 99% pure if it contains less than 1 % contaminating host cell proteins.

The term "polypeptide fragment" as used herein refers to a polypeptide that has a deletion, e.g., an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide, such as a naturally occurring protein. In an embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, or at least 12, 14, 16 or 18 amino acids long, or at least 20 amino acids long, or at least 25, 30, 35, 40 or 45, amino acids, or at least 50 or 60 amino acids long, or at least 70 amino acids long, or at least 100 amino acids long.

The terms "percent identical" or "percent identity," in the context of two or more nucleic acids or polypeptide sequences, refers to the extent to which two or more sequences or subsequences that are the same. Two sequences are "identical" if they have the same sequence of amino acids or nucleotides over the region being compared. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 30 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85 :2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g. , Brent et al., Current Protocols in Molecular Biology, 2003).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403- 410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11 , an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 11 -17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch, J. Mol. Biol. 48:444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.

Other than percentage of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

As used herein a “functional variant” or a given protein includes the wild-type version of said protein, a variant protein belonging to the same family, an homolog protein, or a truncated version, which preserves the functionality of the given protein. Typically, the functional variant exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more amino acid identity with the given protein.

As used herein, the term "mammal" refers to any member of the taxonomic class mammalian, including placental mammals and marsupial mammals. Thus, "mammal" includes humans, primates, livestock, and laboratory mammals. Exemplary mammals include a rodent, a mouse, a rat, a rabbit, a dog, a cat, a sheep, a horse, a goat, a llama, cattle, a primate, a pig, and any other mammal. In some embodiments, the mammal is at least one of a transgenic mammal, a genetically-engineered mammal, and a cloned mammal.

According to the present disclosure, the term "disease" refers to any pathological state, including cancer diseases, in particular those forms of cancer diseases described herein.

The term "normal" refers to the healthy state or the conditions in a healthy subject or tissue, i.e., non-pathological conditions, wherein "healthy" preferably means non-cancerous.

The term "malignancy" refers to a non-benign tumor or a cancer. As used herein, the term "cancer" includes a malignancy characterized by deregulated or uncontrolled cell growth.

The term "cancer" includes primary malignant tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor).

Cancers are classified by the type of cell that resembles the tumor and, therefore, the tissue presumed to be the origin of the tumor. These are the histology and the location, respectively. The term "cancer" according to the disclosure comprises notably leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas and sarcomas. The term cancer notably include rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, cancer of the uterus, ovarian cancer and lung cancer, soft tissue tumors and the metastases thereof. The term cancer according to the present disclosure also comprises cancer metastases and relapse of cancer.

"Growth of a tumor" or "tumor growth" according to the present disclosure relates to the tendency of a tumor to increase its size and/or to the tendency of tumor cells to proliferate.

For purposes of the present disclosure, the terms "cancer" and "cancer disease" are used interchangeably with the terms "tumor" and "tumor disease".

By "treat" is meant to administer a compound or composition as described herein to a subject in order to prevent or eliminate a disease, including reducing the size of a tumor or the number of tumors in a subject; arrest or slow a disease in a subject; inhibit or slow the development of a new disease in a subject; decrease the frequency or severity of symptoms and/or recurrences in a subject who currently has or who previously has had a disease; and/or prolong, i.e. increase the lifespan of the subject. In particular, the term "treatment of a disease" includes curing, shortening the duration, ameliorating, preventing, slowing down or inhibiting progression or worsening, or preventing or delaying the onset of a disease or the symptoms thereof.

The therapeutically active agents or product, vaccines and compositions described herein may be administered via any conventional route, including by injection or infusion.

The agents described herein are administered in effective amounts. An "effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of treatment of a particular disease or of a particular condition, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease or of a condition may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of an agent described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the agents described herein may depend on several of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

The pharmaceutical compositions as herein described are preferably sterile and contain an effective amount of the therapeutically active substance to generate the desired reaction or the desired effect.

The pharmaceutical compositions as herein described are generally administered in pharmaceutically compatible amounts and in pharmaceutically compatible preparation. The term "pharmaceutically compatible" refers to a nontoxic material which does not interact with the action of the active component of the pharmaceutical composition. Preparations of this kind typically contain pharmaceutically compatible excipients such that salts, buffer substances, preservatives, carriers, supplementing immunity-enhancing substances like adjuvants, e.g. CpG oligonucleotides, cytokines, chemokines, saponin, GM-CSF and/or RNA and, where appropriate, other therapeutically active compounds.

Single domain antibodies and variants thereof

As used herein, the term "GPC4” for Glypican 4 has its general meaning in the art and includes human GPC4, in particular the native-sequence polypeptide, isoforms, chimeric polypeptides, all homologs, fragments, and precursors of human GPC4. The amino acid sequence for native GPC4 includes the UniProt reference 075487 (GPC4_HUMAN).

More specifically the term “GPC4” includes the human Glypican 4 (GPC4) of the following SEQ ID: 17: >sp|O75487|GPC4_HUMAN Glypican-4 OS=Homo sapiens OX=9606 GN=GPC4 PE=1 SV=4

The term "antibody", broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1 , lgG2, IgG 3, lgG4, IgAI and lgA2) or subclass. The term "CDR" refers to the complementarity-determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain of immunoglobulins. The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems (such as the Kabat or IM GT numbering).

An antibody fragment is a portion of an antibody, for example as F(ab')2, Fab, Fv, sFv and the like. Functional fragments of a full-length antibody retain the target specificity of a full- length antibody. Recombinant functional antibody fragments, such as Fab (Fragment, antibody), scFv (single chain variable chain fragments) and single domain antibodies (dAbs) have therefore been used to develop therapeutics as an alternative to therapeutics based on mAbs. scFv fragments (~25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domains are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv). The smallest antigen binding fragment is the single variable fragment, namely the VH or VL domain. Binding to a light chain/heavy chain partner respectively is not required for target binding. Such fragments are used in single domain antibodies. A single domain antibody (-12 to 15 kDa) therefore has either the VH or VL domain.

As used herein the term "single-domain antibody" (sdAb) or nanobody® (tradename of Ablynx). has its general meaning in the art and refers to an antibody fragment with a molecular weight of only 12-15 kDa consisting of the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals, and which are naturally devoid of light chains. Thus, in some embodiments, such single-domain antibodies can be VHHs (variable heavy homodimers). For a general description of these (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al, Trends Biotechnol, 2003, 21(1 l):484-490; and WO 06/030220, WO 06/003388. The amino acid sequence and structure of a single-domain antibody can be considered to be comprised of four framework regions or "FRs" which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3 " or "FR3"; and as "Framework region 4" or "FR4" respectively; which framework regions are interrupted by three complementary determining regions or "CDRs", which are referred to in the art as "Complementary Determining Region 1" or "CDR1"; as "Complementarity Determining Region 2" or "CDR2" and as "Complementarity Determining Region 3" or "CDR3", respectively. Accordingly, the single-domain antibody can be defined as an amino acid sequence with the general structure : FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4 respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3. In the context of the present disclosure, the amino acid residues of the single-domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics (IMGT) information system aminoacid numbering (http://imgt.cmes .fr/).

An "isolated sdAb", as used herein, refers to a single domain antibody (sdAb) that is substantially free of other antibodies, notably having different antigenic specificities. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

As used herein, the term "synthetic" means that such antibody has not been obtained from fragments of naturally occurring antibodies but produced from recombinant nucleic acids comprising artificial coding sequences. Accordingly, the single domain antibodies are described herein are typically synthetic single domain antibodies.

As used herein the terms anti-GPC4 antibody or anti-GPC4 sdAb have the same meaning as the terms an antibody, or a sdAb, directed against the GPC4 protein, and notably directed against the human GPC4 protein of SEQ ID NO: 17. sdAb affinity refers to the strength with which the sdAb binds to the epitope presented on an antigen, such as GPC4 in the present disclosure, through its antigen-binding site (paratope). Affinity may be determined based on assessment of the KD value.

The term "KD", as used herein, is intended to refer to the equilibrium dissociation constant, which is obtained from the ratio of k_off to k_on (i.e. koff/k_on) and is expressed as a molar concentration (M). The KD value relates to the concentration of antibody (the amount of antibody needed for a particular experiment) and so the lower the KD value (lower concentration) and thus the higher the affinity of the antibody. KD values for antibodies can be determined using methods well established in the art. Methods for determining the KD values of mAbs can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988 ; Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., 1992, 1993, and Muller, Meth. Enzymol. 92:589-601, 1983, which references are entirely incorporated herein by reference. A method for determining the KD of an antibody is by using surface plasmon resonance, or by using a biosensor system such as a Biacore® (see also for detailed information regarding affinity assessment Rich RL et al., Anal Biochem, 2001, but also for more details about the specific implementation of affinity measurement for sdAb Moutel S et al., eLife 2016;5:el6228). Affinity measurements are generally performed at 25°C. The terms "k_aSsoc" or "ka", or “kon” as used herein, is intended to refer to the association rate of a particular antibodyantigen interaction, whereas the terms "kdis" or "kd,", or koff as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. Briefly, as sdAb are smaller proteins that their respective antigens, they can be capture on a sensorship from a Biocore biosensor instrument, while the recombinant antigens (i.e., typically rGPC4) can be used as analytes. Analytes can be injected sequentially with increased concentration ranging for example between 3.125 nM to 50 nM in a single cycle without regeneration of the sensorship between injections. Binding parameters can be obtained by fitting the overlaid sensorgrams with the 1:1. Langmuir binding model of the BIAevalutation software. Affinity (KD measurement) can also be assessed using Bio-Layer Interferometry measurements (BLItz System instrument (Fortebio)). BLI is an optical analytical technique that analyses the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in realtime. The binding between the ligand immobilized on the biosensor tip surface and the sdAb in solution produces an increase in optical thickness at the biosensor tip, which results in a wavelength shift, AZ, (which is a direct measure of the change in thickness of the biological layer. Interactions are measured in real time, providing the ability to monitor rates of association and dissociation with precision and accuracy. Only molecules binding to or dissociating from the biosensor can shift the interference pattern and generate a response profile on the Octet® System. Unbound molecules, changes in the refractive index of the surrounding medium, or changes in flow rate do not affect the interference pattern. KD assessment using BLi is detailed in the Example Section herein (typically using hGPC4-Fc protein immobilized on a Protein A biosensor)

In some embodiments, the apparent affinity may be assessed in binding assays using an ELISA assay (with typically hGPC4-Fc coated wells) or flow cytometry (typically using HeLa cells expressing recombinant hGPC4), as detailed in the Example section herein.

Typically, a single domain antibody as per the present disclosure binds to GPC4, notably human GPC4 as herein defined with a KD binding affinity of about 10'⁶ M or less, 10'⁷ M or less, 10'⁸ M or less, 10'⁹ M or less, 10'¹⁰ M or less, or 10'¹¹ M or less. Preferably the KD binding affinity is comprised between 10'⁷ and 10'¹⁰ M, notably 10'⁸ and 10'¹⁰, as notably assessed using a binding assay as mentioned above and detailed in the examples section.

In some embodiments, an sdAb according to the present disclosure blocks hGPC4 biological functions, notably in the form of sdAb-Fc format, notably a bivalent sdAb-Fc format, (see below for details), more particularly said sdAb have neutralizing capacities. Typically in some embodiments, treatment of hiPSCs with the an sdAb according to the present invention (in particular in a sdAb-Fc format as detailed below in the Specifications) is able to block 11GPC4 activity and induce differentiation properties, as assessed through a more efficient endoderm lineage entry. The ability of a sdAb-Fc to influence the endoderm differentiation of hiPSCs can be tested by performing differentiation experiments in the presence of various concentrations of an sdAb-Fc or of an irrelevant sdAb, as negative control. sdAb-Fc can typically be applied at the concentrations of 50, 250 and 500nM. The tested sdAb-Fc can be for example applied at the onset of endoderm differentiation and maintained throughout all differentiation procedure, or hiPSCs can be exposed to the tested sdAb-Fc for 24 hours before the start of differentiation and then treated with it during the overall differentiation experiment. Thus typically, treatment of hiPSCs with an sdAb-Fc having neutralizing capacities at undifferentiated stages should result in an hiPSC type wherein functional features are similar to hiPSCs, wherein GPC4 is down-regulated (using for example shRNAs). Entering of hiPSCs into the endodermal lineage can be assessed using SOX17 as a marker. Typically, a GPC4 neutralizing sdAb in an Fc format as detailed herein to promote at least a 10 % increase of SOX17 positive cells in differentiating hiPSCs with respect to hiPSCs treated with the same concentration of an irrelevant sdAb-Fc at a concentration of 500 nM. Preferably also, sdAbs according to the present invention have non-toxic effect when applied to cells at nanomolar concentrations as assessed in a cell survival assay as detailed in the example section, wherein quantification analysis of surviving cells, is typically done by using a metabolic activity-based cell viability assay.

The inventors have isolated 2 reference single-domain antibodies (sdAb)s named hereinafter RB 1 and RB3, and a RB3 variant, with the required properties, notably the required affinity and characterized by following sequences:

Table 1: Full sdAb sequences.

The RB3v sdAb has the same FR1, CDR1, FR2, CDR2, CDR3 and FR4 as RB3. This variant only differs from one amino acid Y58 in RB3 than is changed for N58 in its FR3. Therefore, the present disclosure encompasses single domain antibodies having at least the 3

CDRs of one of the 2 reference single domain antibodies as defined in table 1.

In some embodiments, sdAbs according to the present disclosure include sdAbs having at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100 percent identity with the amino acid sequences as set forth in any one of SEQ ID NO: 15, 16 or 19. sdAb as per the present disclosure notably include anti-GPC4 sdAbs having framework region sequences that have at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100 percent identity with one or more of the sequences SEQ ID NO:7, 8, 9 and 10 or 11, 12, 13 and 14.

In some embodiments of the present invention, the 3CDR regions of anti-GPC4 sdAbs as herein disclosed are 100% identical to the 3 CDR regions of one of the reference sdAbs (hsdAbs) as defined in table 1. Alternatively, in some embodiments, hsdAbs according to the present disclosure may have an amino acid sequence that have been mutated by amino acid deletion, insertion or substitution, yet that have at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100 percent identity in the CDR regions compared with the CDR regions of the sdAb of table 1. Typically, as per the present disclosure, antibodies may have between 1, 2, 3 or 4 amino acid variations (including deletion, insertion or substitution) in one or more CDRs, as compared to the respective CDR sequences of the sdAb of the table 1.

In some embodiments, the single domain antibody of the present disclosure is a mutant variant of one of the reference single domain antibodies of table 1, having the 3 CDR regions 100% identical to the corresponding 3 CDR regions of said reference sdAb, and wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated by amino acid deletion, insertion or substitution in one or more of the FR1, FR2, FR3 and/or FR4 regions, when compared with the corresponding framework regions of the corresponding reference sdAb.

In some embodiments, an sdAb of the present disclosure has one or more amino acid substitutions, deletions, insertions or other modifications compared to SEQ ID NO: 15 or 16, but retains a biological function of the reference single domain antibodies, and notably of the RBI reference sdAb. Biological functions of the reference sdAbs include GPC4 specificity, affinity, in particular in the form of an GPC4-Fc fusion dimer as described below, and neutralizing ability. Said properties and tests to be performed are detailed in the Example section.

Typically, sdAb variants as herein described as well as sdAbs having one or more mutations in the CDRs and/or having different FR regions as compared to the references sdAb and notably as compared to the RB& referenced sdAb exhibit GPC4 neutralizing ability that may be assessed as illustrated in the example section. More particularly said sdAbs or variants thereof according to the present disclosure undergo efficient endoderm lineage entry of hiPSCs as assessed by quantification of SOX17 positive cells. Typically said sdAb and variants thereof retains at least 50% of the RB 1 neutralizing ability for similar concentration.

Modifications may include one or more substitution, deletion or insertion of one or more codons encoding the single domain antibody or polypeptide that results in a change in the amino acid sequence as compared with the sequence of the reference single domain antibody or polypeptide. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

In some embodiments, the modification is a conservative sequence modification. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into single domain antibody as herein described by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine, aliphatic residues I, L, V, and M, cycloalkenyl-associated residues F, H, W, and Y, hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y, negatively charged residues D and E, polar residues C, D, E, H, K, N, Q, R, S, and T, positively charged residues H, K, and R, small residues A, C, D, G, N, P, S, T, and V, very small residues A, G, and S, residues involved in turn A, C, D, E, G, H, K, N, Q, R, S, P, and formation T, flexible residues Q, T, K, S, G, P, D, E, and R.

More conservative substitution groupings include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservation in terms of hydropathic/hydrophilic properties and residue weight/size also is substantially retained in a variant as compared to a CDR of the any one of mAbs 1-11. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (- 0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The retention of similar residues may also or alternatively be measured by a similarity score, as determined by use of a BLAST program (e.g., BLAST 2.2.8 available through the NCBI using standard settings BLOSUM62, Open Gap= 11 and Extended Gap= 1). In some embodiments, the variants has the 6 CDR regions 100% identical to the corresponding 6 CDR regions of the reference mAb, and include mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated by amino acid deletion, insertion or substitution, preferably bu conservative substitution, in the FR1, FR2, FR3 and FR4 regions when compared with the corresponding framework regions of the corresponding reference antibody.

In some embodiments, the single domain antibody is selected from one of SEQ ID NO: 15 or 16, but comprises one or more amino acid substitutions, for example 1 to 20, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions. The one or more amino acid substitution can be in one or more of the framework areas. Alternatively, or in addition, the one or more amino acid substitution can be in one or more of the CDRs. In some embodiments, the amino acid substitutions are in the framework and CDR sequences. The present disclosure notably include sdAbs having 3 CDRs of SEQ ID NO: 1-3 or 4-6 and wherein one or more CDRs, include one or more (typically 1 or 2) conservative amino acid modifications, and in particular one or more (typically 1 or 2) conservative substitution as above defined. The variant sdAb can be tested for retained function notably by using the functional assays described herein (notably the binding assay detailed in the Example section).

In some embodiments, the single domain antibody is a variant of a single domain antibody selected from those having SEQ ID NO: 15, 16 or 19, that comprises one or more sequence modification and has improvements in one or more of a property such as binding affinity, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility as compared to the unmodified single domain antibody.

A skilled person will know that there are different ways to identify, obtain and optimise the antigen binding molecules as described herein, including in vitro and in vivo expression libraries. Optimisation techniques known in the art, such as display (e.g., ribosome and/or phage display) and / or mutagenesis (e.g., error-prone mutagenesis) can be used. The present disclosure therefore also comprises sequence optimised variants of the single domain antibodies described herein.

The present disclosure also includes humanized format of anti-GPC4 sdAbs as herein disclosed. Typically, such humanized anti-GPC4 sdAbs have CDRs that are identical to the ones of the 2 reference single domain antibodies as defined in table 1 (i.e: CDR1 of SEQ ID NO1:, CDR2 of SEQ ID NO2: and CDR3 of SEQ ID NO3; or CDR1 of SEQ ID NO4:, CDR2 of SEQ ID NO5: and CDR3 of SEQ ID NO:6).

In some embodiments, humanization may require that one or more substitutions, notably a conservative amino acid modification, and in particular a conservative amino acid substitution (see above for details) may be included in one or more of the CDRs as above mentioned.

In some embodiments, humanized anti-GPC4 antibody have a sequence having at least 90 %, notably at least 95, 96, 97, 98, 99 or more percent identity with the amino acid sequences as set forth in any one of SEQ ID NO: 15, 16 or 19.

Typically, the sdAb variants as defined above, of the reference RB I, RB3 or RB3v sdAb disclosed herein, retain the function of their respective parent sdAb (i.e.: RB I, RB3, or RB3v).

In particular, said sdAb variants bind to GPC4, notably human GPC4 as herein defined, with a KD binding affinity of about 10'⁶ M or less, 10'⁷ M or less, 10'⁸ M or less, 10'⁹ M or less, 10'¹⁰ M or less, or 10'¹¹ M or less. Preferably the KD binding affinity is comprised between 10’ ⁷ and 10'¹⁰ M, notably 10'⁸ and 10'¹⁰, as notably assessed using a binding assay as mentioned above and detailed in the examples section. More particularly such sdAb variants block 11GPC4 biological functions, notably in the form of a sdAb-Fc (see below for details), more particularly said sdAb have neutralizing capacities as previously defined.

Single domain antibody engineering

In some embodiments, sdAbs according to the present disclosure can be chemically modified, for example to increase their molecular weight to reduce renal clearance or protect for example from proteases. PEGylation (covalent attachment of a polyethylene glycol (PEG) group) for example has been widely used to increase the half-life. Other strategies to limit renal clearance involve attachment of negative charges to the sdAb, such as addition of sialic acid polymers (polysialylation) or hydroxyethal starch (HESylation) and by fusion with the highly syaliated beta carboxyterminal peptide (CTP) amino acid-residue of the human chorionic gonadotrophin (hCG) hormone.

In some embodiments of the present disclosure, an isolated single domain antibody as herein described can also be linked directly or not (i.e. typically vi an adapted linker), covalently or not to a compound of interest. The substance or compound of interest as defined above can be directly and covalently, or non-covalently, linked to a single domain antibody as herein defined either to one of the terminal ends (N or C terminus), or to the side chain of one of the amino acids of said single domain antibody. The substance of interest can thus be indirectly and covalently or non-covalently linked to said single domain antibody by means of a spacer either to one of the terminal ends of said single domain antibody, or to a side chain of one of the amino acids of said single domain antibody.

Conventional linking methods of a substance of interest to a peptide, in particular an antibody, are known in the art (e.g., See Temynck and Avrameas 1987 "Techniques immunoenzymatiques" Ed. INSERM, Paris; Hermanson, 2010, Bioconjugate Techniques, Academic Press).

In some embodiments, single domain antibodies as herein described can be notably in the form of “antibody drug conjugate” of the formula sdAb-(L- (D)m)n or a pharmaceutically acceptable salt thereof; wherein sdAb is a single domain antibody as previously disclosed; L is a linker; D is a compound of interest; m is an integer from 1 to 8; and n is an integer from 1 to 10, typically 3 or 4. The term "antibody drug conjugate" (ADC) as used herein refers to the linkage of a single domain antibody with another agent, or compound of interest, such as for example a chemotherapeutic agent, a toxin, a polypeptide or a protein, an immunotherapeutic agent, an imaging probe, and the like. The linkage can be covalent bonds, or non-covalent interactions such as through electrostatic forces. Various linkers, known in the art, can be employed in order to form the immunoconjugate. The linker (L) can be for example selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a procharged linker and a dicarboxylic acid-based linker.

In some embodiments, the single domain antibody of the present disclosure is conjugated, or covalently linked to the compound of interest. As used herein, the term "conjugation" has its general meaning in the art and means a chemical conjugation, or chemical crosslinking. Many chemical cross-linking methods are also known in the art. Cross-linking reagents may be homobifunctional (i.e., having two functional groups that undergo the same reaction) or heterobifunctional (i.e., having two different functional groups). Numerous crosslinking reagents are commercially available. Detailed instructions for their use are readily available from the commercial suppliers. A general reference on polypeptide cross-linking and conjugate preparation is: WONG, Chemistry of protein conjugation and cross-linking, CRC Press (1991), see also Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker, Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62: 119-58. See also, e.g., PCT publication WO 89/12624.). Typically, the nucleic acid molecule is covalently attached to lysines or cysteines on the antibody, through N-hydroxysuccinimide ester or maleimide functionality respectively. Methods of conjugation using engineered cysteines or incorporation of unnatural amino acids have been reported to improve the homogeneity of the conjugate (Axup, J.Y., Bajjuri, K.M., Ritland, M., Hutchins, B.M., Kim, C.H., Kazane, S.A., Haider, R., Forsyth, J.S., Santidrian, A.F., Stafin, K., et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101-16106.; Junutula, J.R., Flagella, K.M., Graham, R.A., Parsons, K.L., Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, D.L., Li, G., et al. (2010) Engineered thio-trastuzumab-DMl conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin. Cancer Res.16, 4769-4778.). Junutula et al. (2008) developed cysteine-based site-specific conjugation called "THIOMABs" (TDCs) that are claimed to display an improved therapeutic index as compared to conventional conjugation methods. In some embodiments, the single domain antibody of the present disclosure is conjugated to the heterologous moiety by a linker molecule. As used herein, the term "linker molecule" refers to any molecule attached to the single domain antibody the present disclosure. The attachment is typically covalent. In some embodiments, the linker molecule is flexible and does not interfere with the binding of the single domain antibody the present disclosure.

A compound or substance of interest as herein intended can be selected without limitation from a nucleic acid, a polypeptide or a protein, a virus, a bacteria, a toxin and a chemical entity.

The compound or substance of interest, as above described, can be a therapeutic or a diagnostic compound. Therapeutic compounds notably include therapeutic compounds having anti-cancer and/or cytotoxic activity, or anti-viral activity and diagnostic compounds such as typically imaging probes.

In more specific embodiments, said substance of interest is a lipoparticle or a polymeric entity comprising, or encapsulating a diagnostic or therapeutic compound (Villaraza et al. 2010 Chem Rev., 110, 2921-2959). Hence, nanobodies are very convenient tools for delivering toxic cargos to cancer cells and are well-suited for chemical conjugation onto different nanoparticle formats. Carrier (or cargos) may include lipoparticles such as liposomes or micelles, albuminbased nanoparticles and polymer-based polymersomes.

The term "toxin," "cytotoxin" or "cytotoxic compound" as used herein, refers to any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit, or destroy a cell or malignancy.

The term "anti-cancer compound" as used herein refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to, cytotoxic agents, chemotherapeutic agents, radioisotopes, targeted anti-cancer agents, immunotherapeutic agents (such as immunosuppressants or immune stimulators), and lytic peptides A therapeutic compound having anti-cancer or cytotoxic activity can be for example selected from a group consisting of a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2 inhibitor, an MCL1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1 , a DPPIV inhibitor, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HD AC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.

In some embodiments, the single domain antibody is conjugated to a cytotoxic moiety. The cytotoxic moiety may, for example, be selected from the group consisting of taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxyanthracindione; a tubulin-inhibitor such as maytansine or an analog or derivative thereof; an antimitotic agent such as mo no methyl auristatin E or F or an analog or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1 -dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analog or derivative thereof; an antimetabolite such as methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabin, 5 fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; an alkylating agent such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; a platinum derivative such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), or an analog or derivative thereof; an antibiotic such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)); pyrrolo[2,l-c][l,4]- benzodiazepines (PDB); diphtheria toxin and related molecules such as diphtheria A chain and active fragments thereof and hybrid molecules, ricin toxin such as ricin A or a deglycosylated ricin A chain toxin, cholera toxin, a Shiga- like toxin such as SLT I, SLT II, SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaccaamericana proteins such as PAPI, PAPII, and PAP-S, momordicacharantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, pheno mycin, and enomycin toxins; ribonuclease (R ase); DNase I, Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtherin toxin; and Pseudomonas endotoxin.

In some embodiments, the single domain antibody is conjugated to an auristatin or a peptide analog, derivative or prodrug thereof. Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anti-cancer (US5663149) and antifungal activity (Pettit et al, (1998) Antimicrob. Agents and Chemother. 42: 2961-2965). For example, auristatin E can be reacted with para-acetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP, MMAF (monomethylauristatin F), and MMAE (monomethylauristatin E). Suitable auristatins and auristatinanalogs, derivatives and prodrugs, as well as suitable linkers for conjugation of auristatins to Abs, are described in, e.g., U.S. Patent Nos. 5,635,483, 5,780,588 and 6,214,345 and in International patent application publications W002088172, W02004010957, W02005081711, W02005084390, W02006132670, WO03026577, W0200700860, W0207011968 and W0205082023.

In some embodiments, the single domain antibody is conjugated to Mertansine (also called emtansine or DM1) or a peptide analog, derivative or prodrug thereof. Mertansine is a tubulin inhibitor, meaning that it inhibits the assembly of microtubules by binding to tubulin.

In some embodiments, the single domain antibody is conjugated to pyrrolo[2,l-c][l,4]- benzodiazepine (PDB) or an analog, derivative or prodrug thereof. Suitable PDBs and PDB derivatives, and related technologies are described in, e.g., Hartley J. A. et al, Cancer Res 2010; 70(17) : 6849-6858; Antonow D. et al, Cancer J 2008; 14(3) : 154-169; Howard P.W. et al, Bioorg Med ChemLett 2009; 19: 6463-6466 and Sagnou et al, Bioorg Med ChemLett 2000; 10(18) : 2083-2086.

In some embodiments, the single domain antibody is conjugated to a cytotoxic moiety selected from the group consisting of an anthracycline, maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethylauristatin E, monomethylauristatin F, a PDB, or an analog, derivative, or prodrug of any thereof.

In some embodiments, the single domain antibody is conjugated to an anthracycline or an analog, derivative or prodrug thereof. In some embodiments, the single domain antibody is conjugated to maytansine or an analog, derivative or prodrug thereof. In some embodiments, the single domain antibody is conjugated to calicheamicin or an analog, derivative or prodrug thereof. In some embodiments, the single domain antibody is conjugated to duocarmycin or an analog, derivative or prodrug thereof. In some embodiments, the single domain antibody is conjugated to rachelmycin (CC-1065) or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 10 or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 15 or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to monomethylauristatin E or an analog, derivative or prodrug thereof. In some embodiments, the single domain antibody is conjugated to monomethylauristatin F or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to pyrrolo[2,l-c][l,4]- benzodiazepine or an analog, derivative or prodrug thereof. In some embodiments, the single domain antibody is conjugated to irinotecan or an analog, derivative or prodrug thereof.

In some embodiments, the sdAb is conjugated to a nucleic acid or nucleic acid- associated molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease (RNase) or deoxy-ribonuclease (e.g., DNase I), an antisense nucleic acid, an inhibitory RNA molecule (e.g., a siRNA molecule) or an immuno stimulatory nucleic acid (e.g., an immunostimulatoryCpG motif-containing DNA molecule). In some embodiments, the antibody is conjugated to an aptamer or a ribozyme.

In some embodiments, the sdAb is conjugated, e.g., as a fusion protein, to a lytic peptide such as CLIP, Magainin 2, mellitin, Cecropin and PI 8.

In some embodiments, the single domain antibody is conjugated to a cytokine, such as, e.g., IL-2, IL- 4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL- 28b, IL-29, KGF, IFNa, IFN3, IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa.

In some embodiments, the single domain antibody is conjugated to a radioisotope or to a radioisotope-containing chelate. For example, the antibody can be conjugated to a chelator linker, e.g. DOTA, DTPA or tiuxetan, which allows for the antibody to be complexed with a radioisotope. The single domain antibody may also or alternatively comprise or be conjugated to one or more radiolabeled amino acids or other radiolabeled moleculesNon- limiting examples of radioisotopes include ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, "Tc, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ²¹³Bi, ²²⁵ Ac and ²²⁷Th. For therapeutic purposes, a radioisotope emitting beta- or alpha-particle radiation can be used, e.g.

1311, 90Y, 211At, 212Bi, 67Cu, 186Re, 188Re, and 212Pb.

A diagnostic compound can be selected from an enzyme, a fluorophore, a NMR or MRI contrast agent, a radioisotope or a nanoparticle. For example, the diagnostic compound can be selected from the group consisting of:

- an enzyme such as horseradish peroxidase, alkaline phosphatase, glucose-6- phosphatase or beta-galactosidase;

- a fluorophore such as green fluorescent protein (GFP), blue fluorescent dyes excited at wavelengths in the ultraviolet (UV) part of the spectrum (e.g. AMCA (7-amino-4- methylcournarin-3 -acetic acid); Alexa Fluor® 350), green fluorescent dyes excited by blue light (e.g. FITC, Cy2, Alexa Fluor® 488), red fluorescent dyes excited by green light (e.g. rhodamines, Texas Red, Cy3, Alexa Fluor dyes 546, 564 and 594), or dyes excited with far-red light (e.g. Cy5) to be visualized with electronic detectors (CCD cameras, photomultipliers);

- a radioisotope such as 18F, nC, 13N, 150, 68Ga, 82Rb, 44Sc, 64Cu, 86Y, 89Zr, 1241, 152Tb that can be used for PET imaging or 67Ga, 81mKr, 99mTc, mln, 1231, 1251, ,3 Xe, 201T1, 155Tb, 195mPt that can be used for SPECT / scintigraphic studies, or 14C, 3H, 35S, 3 P, 1251 that can be 211 212 75 76 131 1 1 1 used for autoradiography or in situ hybridisation, or At-, Bi-, Br-, Br-, I-, In, 177Lu-, 212Pb-, 186Re-, 188Re-, 153Sm-, 0Y that can be used to label the compounds;

- a NMR or MRI contrast agent such as the paramagnetic agents gadolinium (Gd), dysprosium (Dy) and manganese (Mn), and the superparamagnetic agents based on iron oxide (such as MION, SPIO or USPIO) or iron platinium (SIPP), and X-nuclei such as 18F, 13C, 23Na, 170, 15N;

- a nanoparticle such as gold nanoparticles (B. Van de Broek et al, ACSNano, Vol. 5, No. 6, 4319-4328, 2011) or quantum dots (A. Sukhanova et al, 2012 Nanomedicine, 8 516- 525).

In a preferred embodiment, said diagnostic compound is a fluorophore, more preferably Alexa Fluor® 488, or a MRI contrast agent, more preferably gadolinium.

When the diagnostic agent is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example 99Tc or 1231, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as MRI), such as 13C, 9F, Fe, Gd, 1231, n Un, Mn, 15N or 70. The substance of interest according to the present disclosure may or may not permeate the mammal or human blood-brain barrier.

In some embodiments, when the compound of interest is a heterologous or a homologous polypeptide, the single domain antibody of the present disclosure can be (alternatively, or in addition) fused (more generally using a linker or spacer as previously described) to one or more heterologous/homologous polypeptide(s) or to a protein to form a fusion protein (also named herein “fusion polypeptide” or “polypeptide”). A "fusion" or "chimeric" protein or polypeptide comprises a first amino acid sequence linked (usually by using a linker or spacer as previously described) to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences, which normally exist in separate proteins can be brought together in the fusion polypeptide. A fusion protein or polypeptide is created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the polypeptide regions are encoded in the desired relationship.

According to the present disclosure, the fusion protein can thus comprise at least one isolated single domain antibody (hsbAb) according to the present disclosure that is fused either directly or via a linker or spacer at its C-terminal end and / or at its N terminal end, notably fused at its C-terminal end to the N-terminal end of the heterologous polypeptide, and/ or at its N-terminal end to the C- terminal end of the heterologous polypeptide. As used herein, the term "directly" means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the humanized single domain antibody is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of the heterologous polypeptide. In other words, in this embodiment, the last amino acid of the C-terminal end of said sdAb is directly linked by a covalent bond to the first amino acid of the N- terminal end of said heterologous polypeptide, or the first amino acid of the N-terminal end of said sdAb is directly linked by a covalent bond to the last amino acid of the C-terminal end of said heterologous polypeptide. As used herein, the term "spacer" also called "linker" refers to a sequence of at least one amino acid that links the sdAb of the present disclosure to the heterologous polypeptide (spacer or linkers have also been described previously in relation with conjugation techniques). Such a spacer may be useful to prevent steric hindrances. Examples of linkers disclosed in the present disclosure have the following sequences (Gly3-Ser)4, (Gly3-Ser), Ser-Gly or (Ala- Ala- Ala). Appropriate linkers include flexible Gly/Ser based polypeptide linkers comprising between about 5 to 40 amino acids, notably of 5 to 25 or about 10 to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, but linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline may also be be suitable. A well-suited linker according to the present disclosure contains glycine and serine residues and is for example of the format (GGGGS)p, with p is an integer comprised between 1 and 8, notably between 1 and 4, notably p is 2; 3 or 4. Suitable linker examples also include 9GS [(Gly)4Ser(Gly)3Ser], 12GS [(Gly)3(Ser)]3, and 30GS [(GlyMScrJJe linkers.

In some embodiments, the polypeptide or protein, can be an enzyme, such as a reporter enzyme, albumin, or an immunoglobulin.

In some embodiments, the compound of interest can also be one or more polypeptides comprising another or the same antigen binding domain to form a multivalent binding compound. Notably, the compound of interest can be one or more single domain antibodies as herein disclosed or not. The resulting fusion protein, or polypeptide, that comprises two or more antigen binding domains, notably that comprises or essentially consists of two or more single domain antibodies, are referred to herein as "multivalent" polypeptides or (multivalent) antigen binding compounds. In some embodiments, said fusion protein or polypeptide can comprise at least one single domain antibody with a first binding domain, as herein described, and at least one other binding domain (e.g. directed against the same or another epitope, antigen, target, protein or polypeptide), which is typically also a single domain antibody. "Multispecific" (fusion) polypeptide refers to a polypeptide comprising at least two different antigen binding domains (i.e. that target different epitopes, antigens or targets), in opposition to a polypeptide comprising similar antigen binding domains, notably comprising the same single domain antibodies (multivalent "monospecific" (fusion) polypeptides). Thus, the present disclosure encompasses multivalent binding compounds comprising at least two single domain antibodies as herein disclosed (for example two RB 1 sdAbs or variants thereof) build in a tandem format, notably a head to tail tandem format.

In some embodiments, a fusion protein as herein described may also comprise at least a second antigen binding domain directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope. Said binding domain can be directed against GPC4, notably against the same or different GPC4 epitope, or may be directed against any other antigen, polypeptide or protein.

A "bispecific" fusion protein or bispecific binding compound of the present disclosure is a fusion polypeptide that comprises at least one single domain antibody as herein disclosed directed against a first antigen (i.e. GPC4) and at least one further binding domain directed against a second GPC4 epitope or a different target or antigen, whereas a "trispecific" polypeptide or binding compound of the present disclosure is a polypeptide that comprises at least one single domain antibody as herein disclosed and directed against a first antigen (i.e. GPC4), at least one further binding domain directed against a second GPC4 epitope or antigen (i.e. different from GPC4) and at least one further binding domain directed against a third GPC4 epitope or antigen (i.e. different from both i.e. first and second antigen); etc.

Typically antigens other than GPC4 can be selected from PSMA, PSCA, BCMA, CS1 , GPC3, CSPG4, EGFR, HER3, CA125, CD123, 5T4, IL-13R, CD2, CD3, CD16 (FcyRIII), CD23, LI CAM, MUC16, ROR1 , SLAMF7, cKit, CD38, CD53, CD71, CD74, CD92, CD100, CD123, CD138, CD146 (MUC18), CD148, CD150, CD200, CD261, CD262, CD362, ROR1 , mesothelin, CD33/IL3Ra, c-Met, Glycolipid F77, EGFRvlll, MART-1, gplOO, GD-2, O-GD2, NKp46 receptor, or presented antigens like NY-ESO-1 or MAGE A3, human telomerase reverse transcriptase (hTERT), survivin, cytochrome P450 1 B l (CY1 B), Wilm's tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16, MUC1 , p53, cyclin, an immune checkpoint target or combinations thereof. In some embodiments the antigen can also be Igg or albumin antigens.

In some embodiment, the at least one further antigen of the multispecific fusion polypeptide, or binding compound, comprises at least an immune cell antigen such as one or more T cell antigens, one or more macrophage antigens, one or more NK cell antigens, one or more neutrophil antigens, and/or one or more eosinophil antigens, as typically exemplifier for Bispecific T-cell or NK-cell engager molecules (see notably for BiTEs® Wolf E, Hofmeister R, Kufer P, Schlereth B, Baeuerle PA. “BiTEs: bispecific antibody constructs with unique antitumor activity”. Drug Discov Today. 2005 Sep 15;10(18): 1237-44. Review). Amongst others for T cell antigens, CD2 and framework sequences of T-cell receptor a and P chains can be used, notably CD2 or CD3 and most particularly the e chain of the CD3 complex. For example, for NK cell antigens fragments from the FcyRIII and/or from the NKp46 receptor can be used.

Said multispecific polypeptides can be used immune cell redirecting immune therapies on the same principle as for CAR therapies (see for illustrative review Ellwanger K, Reusch U, Fucek I, et al. Redirected optimized cell killing (ROCK®): A highly versatile multispecific fit- for-purpose antibody platform for engaging innate immunity. MAbs. 2019; 11(5):899— 918). In some embodiments, a further binding domain can be directed against a serum protein so that the half-life of the single domain antibody is increased. Typically, said serum protein is albumin.

In some embodiments, a further binding domain can be directed against a receptor on the vascular endothelium of the blood-brain barrier so that the single domain antibodies of the present disclosure would cross the blood-brain barrier. The targeted receptors include transferrin receptor, insulin receptor, IGF-I and IGF-II receptors, among others.

In some embodiments, the one or more further binding domain may comprise one or more parts, fragments or domains of conventional chain antibodies (and in particular human antibodies) and/or of heavy chain antibodies. For example, a single domain antibody as herein defined may be linked to a conventional (typically human) VH or VL optionally via a linker sequence.

In some embodiments, the polypeptides, or fusion proteins of the present disclosure comprise a single domain antibody of the present disclosure that is linked to an immunoglobulin or a portion or fragment thereof. For example, the polypeptide, or fusion protein comprises a single domain antibody of the present disclosure that is linked to an Fc domain (CH2-CH3), notably a human Fc region. Fc region from various mammals (typically from human or mouse antibodies) antibody subclasses can be used. Said Fc domain may be useful for increasing the half-life and even the production of the single domain antibody of the present disclosure. For example, the Fc portion can bind to serum proteins and thus increases the half-life on the single domain antibody. Fc region also enhance target antigen binding through an avidity effect. The fusion of single domain antibodies to the fragment crystallisable region (Fc-fragment) of conventional antibodies has further been shown to enhance sdAbs potencies and stability in biological fluids as well as endow them with properties such as translocation across the bloodbrain barrier and low cytotoxicity, which could be highly useful in therapy targeting the brain such as the treatment of neurodegenerative disease or brain cancers.

In some embodiments, a single domain antibody as herein disclosed may be linked to one or more (typically human) Hinge and/or CHI, and/or CH2 and/or CH3 domains, optionally via a linker sequence. For instance, a single domain antibody may be linked to a suitable CHI domain and could for example be used - together with suitable light chains - to generate antibody fragments/structures analogous to conventional Fab fragments or F(ab')2 fragments, but in which one or (in case of an F(ab')2 fragment) both of the conventional VH domains have been replaced by a single domain antibody as herein defined.

Typically, one or more single domain antibodies of the present disclosure may be linked (optionally via a suitable linker or hinge region) to one or more constant domains (for example, 2 or 3 constant domains that can be used as part of/to form an Fc portion), to an Fc portion and/or to one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the present disclosure and/or may confer the ability to bind to one or more Fc receptors. For example, for this purpose, and without being limited thereto, the one or more further amino acid sequences may comprise one or more CH2 and/or CH3 domains of an antibody, such as from a heavy chain antibody and more typically from a conventional human chain antibody; and/or may form and Fc region, for example from an IgG (e.g. from IgGl, IgG2, IgG3 or IgG4), an IgE or from another human Ig such as IgA, IgD or IgM. Of course, such formats are also suitable with multivalent and/or multispecific binding domain as above mentioned, wherein at least one sdAb as herein disclosed is used. Thus in some embodiments, the present disclosure encompasses multivalent binding compounds comprising at least two binding domains. Typically, at least one of said binding domain comprises an anti- GPC4 sdAb as herein disclosed. In more specific embodiments, the multivalent binding compound or polypeptide comprises 2 binding domains, wherein at least one and preferably the two binding domains are anti-GPC4 sdAbs as herein disclosed, notably DB 1 sdAbs or variants thereof. In such embodiments, the sdAb can further be linked to an immunoglobulin domain, notably an Fc domain, via its N or C terminal portion as typically illustrated in Figure 2A. More specifically, single domain antibody-based bivalent antibodies can also be obtained by pairing two single-domain antibodies each fused to an Fc fragment (typically comprising CH2 and CH3 constant domains from a human IgG notably from a human IgGl or a human IgG4) to obtain as sdAb-Fc format, notably a bivalent sdAb-Fc format (as typically illustrated in Figure 2).. The sdAb can be fused to the N terminal or the C terminal region of the Fc portion, but typically, the sdAb is fused to the N terminal region of the Fc portion (typically the CH2 constant region) via a linker as above described. The bivalent antibody is typically obtained by pairing the Fc fragments. Thus typically, such bivalent binding compound as the following format (sdAb-L- CH2CH3)2, with L being a linker as herein described. As above mentioned, the 1^st and 2^nd single domain antibodies can target the same or different antigen. In some specific embodiments the 1^st sdAb and 2^nd sdAb are anti-GPC4 antibodies as herein described. Typically, 2 RB I sdAbs of the present disclosure, or variant thereof can be used. Various embodiments have been described to produce single domain antibody -based bivalent antibodies such as in Ridgway JB, Presta LG, Carter P. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization (Protein Eng. 1996;9(7):617- 621); See also Danquah et al. Nanobodies that block gating of the P2X7 ion channel ameliorate inflammation (Sci. Transl. Med.2016 : 8, 366ral62); and Albert et al. From mono- to bivalent: improving theranostic properties of target modules for redirection of UniCAR T cells against EGFR-expressing tumor cells in vitro and in vivo (Oncotarget. 2018; 9:25597-25616). The single domain antibody can be fused (chemically or genetically) to the Fc portion. Fc formatted nanobodies have also been described in Bannas P, Hambach J, Koch-Nolte F. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Front Immunol. 2017 ;8: 1603. Published 2017 Nov 22.; or in Bobkov V, Zarca AM, Van Hout A, Arimont M, Doijen J, Bialkowska M, Toffoli E, Klarenbeek A, van der Woning B, van der Vliet HJ, Van Loy T, de Haard H, Schols D, Heukers R, Smit MJ. Nanobody-Fc constructs targeting chemokine receptor CXCR4 potently inhibit signaling and CXCR4-mediated HIV-entry and induce antibody effector functions. Biochem Pharmacol. 2018 Dec;158:413-424; but see also for general review Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019 Aug;18(8):585-608. Typically, sdAbs can be constructed into bivalent Nb-Fc formats by genetically cloning them in frame with the second and third constant domains (CH2-CH3) of a human IgG (typically IgGl) heavy chain. The resulted constudt can be cloned in an expression vector, and the SdAbs- Fc recovered by purification as classically done in the field. As above mentioned, sdAbs are classically fused to the Fc portion via a flexible linker.

Chimeric antigen receptors

The terms "Chimeric antigen receptor" or "CAR" or "CARs" as used herein refer to engineered receptors, which graft an antigen specificity onto cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combination thereof) thus combining the antigen binding properties of the antigen binding domain with the lytic capacity and self-renewal of T cells. CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors. The term "antigen binding domain or "antigen- specific targeting domain" as used herein refers to the region of the CAR which targets and binds to specific antigens. When a CAR is expressed in a host cell, this domain forms the extracellular domain (ectodomain). The CAR of the present disclosure comprises a molecule of the general formula: sdAb(n)- [optionally a hinge] transmembrane domain- intracellular signaling domain, wherein n is 1 or more.

In some embodiments, n is at least 2, for example 2, 3, 4 or 5. The sdAb(n) form the antigen binding domain and is/are located at the extracellular side when expressed in a cell.

Typically, a CAR as herein described preferably comprises at least two antigen binding domains (each comprising a single domain antibody), which target one or more antigen. The antigen binding domain of a CAR of the present disclosure can comprise two or at least two sdAb that are both specific for the GPC4, thus providing a bivalent binding molecule. In some embodiments, the antigen binding domain comprises two or at least two VH single domain antibodies that are both specific for GPC4 but bind to different epitopes. In other words, the antigen binding domain comprises a first single domain antibody that binds to a first epitope of GPC4 and a second single domain antibody that binds to a second epitope of GPC4. The epitopes may be overlapping. Thus, the antigen binding domain is biparatopic. In other embodiments, the antigen binding domain comprises two single domain antibodies that are both specific for GPC4 and bind to the same epitope.

In some embodiments, the antigen binding domain comprises one single domain antibody according to the present disclosure and that is thus specific for GPC4 and another antigen binding domain that is specific for another antigen, thus providing a bispecific antigen binding domain. In other words, the antigen binding domain comprises a first single domain antibody that binds to a first target consisting in GPC4 and a second single domain antibody that binds to a second target. Thus, in certain embodiments, the present disclosure relates to bispecific CARs.

As used herein, the term "bispecific CAR" or "bispecifc antigen binding domain" thus refers to a polypeptide that has specificity for two targets including GPC4. Accordingly, a bispecific binding molecule as described herein can selectively and specifically bind to a cell that expresses (or displays on its cell surface) GPC4 and the second target.

In other embodiments, the binding molecule comprises more than two antigen-binding domains providing a multispecific binding molecule. A multispecific antigen-binding domain as described herein can thus in addition to binding GPC4 bind one or more additional targets, i.e., a multispecific polypeptide can bind at least two, at least three, at least four, at least five, at least six, or more targets, wherein the multispecific polypeptide agent has at least two, at least, at least three, at least four, at least five, at least six, or more target binding sites respectively.

In some embodiments, additional antigens that can be bound by a multispecific CAR according to the present disclosure include tumor antigens. In some embodiments, the tumor antigens are associated with a hematologic malignancy or with a solid tumor. For example, a tumor antigen can be selected from the group consisting of CD19, CD20, CD33, PSMA, PSCA, BCMA, CS1 , GPC3, CSPG4, EGFR, HER3, CA125, CD123, 5T4, IL-13R, CD2, CD3, CD16 (FcyRIII), CD23, LI CAM, MUC16, ROR1 , SLAMF7, cKit, CD38, CD53, CD71, CD74, CD92, CD100, CD123, CD138, CD146 (MUC18), CD148, CD150, CD200, CD261, CD262, CD362, ROR1 , mesothelin, CD33/IL3Ra, c-Met, Glycolipid F77, EGFRvlll, MART-1, gplOO, GD-2, O-GD2, NKp46 receptor, presented antigens like NY-ESO-1 or MAGE A3, human telomerase reverse transcriptase (hTERT), survivin, cytochrome P450 1 B l (CY1 B), Wilm's tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16, MUC1 , p53, cyclin, and an immune checkpoint target or combinations thereof. However, a skilled person would understand that other tumor antigens are also targets within the scope of the present disclosure.

In addition to a binding domain as described in detail above, a CAR of the present disclosure further comprises a transmembrane domain. A "transmembrane domain" (TMD) as used herein refers to the region of the CAR which crosses the plasma membrane and is connected to the endoplasmic signaling domain and the antigen binding domain, in case of the latter optionally via a hinge. In one embodiment, the transmembrane domain of the CAR of the present disclosure is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof. In some embodiments, the transmembrane domain comprises the CD8 domain, the CD3zeta domain, or the CD28 transmembrane domain. Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the present disclosure.

A CAR of the present disclosure further comprises an intracellular signaling domain. An "intracellular signaling domain", "cytoplasmic domain" or "endodomain" is the domain that transmits activation signals to T cells and directs the cell to perform its specialized function. Examples of domains that transduce the effector function signal and can be used according to the present disclosure include but are not limited to the C, chain of the T-cell receptor complex or any of its homologs (e.g., r| chain, FcsRIy and P chains, MB 1 (Iga) chain, B29 (Ig ) chain, etc.), human CD3zeta chain, CD3 polypeptides (A, 6 and a), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.) and intracellular domains from other molecules involved in T-cell transduction, such as CD2, CD5, 0X40, and CD28. Other intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the present disclosure. In some embodiments, the intracellular domain in notably selected from the intracellular domain from CD28 and the human CD3zeta chain.

Preferably, the CAR comprises additional activation domain(s) (or intracellular domain) comprising a fragment of at least 50, 60, 70, 80, 90,100, 1 10, 120, 150, or 200 amino acids of at least one additional activation domain selected from CD3-^ chain (also shortly named Q and the cytoplasmic domain of a costimulatory receptors CD28, 4-1 BB (CD137), 0X40 (CD134), LAG3, TRIM, HVEM, ICOS, CD27, or CD40L. In various embodiments, the CAR comprises additional activation domain(s) comprising a fragment of at least 20, 30, 40, 50, 60, 70, 80, 90,100, 1 10, 120, 150, or 200 amino acids that shares at least than 90%, preferably more than 95%, more preferably more than 99% identity with the amino acid sequence of the additional activation domain above mentioned.

In some embodiments, a CAR of the present disclosure further comprises one or more co- stimulatory domains to enhance CAR-T cell activity after antigen specific engagement. Inclusion of this domain in the CAR of the present disclosure enhances the proliferation, survival and/or development of memory cells. The co- stimulatory domain is located intracellular^A. The co- stimulatory domain is a functional signaling domain obtained from a protein selected form the following group: CD3zeta, CD28, CD137 (4-IBB), CD134 (0X40), DapIO, CD27, CD2, CD5, ICAM-1 , LFA-1 (CD1 la/CD18), Lek, TNFR-I, TNFR-II, Fas, CD30, CD40, LAG3, TRIM, HVEM, ICOS, CD40L or combinations thereof. Other costimulatory domains (e.g., from other proteins) will be apparent to those of skill in the art. Multiple co- stimulatory domains can be included in a single CAR to recruit multiple signaling pathways. In one embodiment, the co-stimulatory domain is obtained from 4-1 BB. The term "4-1 BB" refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non- human species, e.g., rodent (e.g. mouse or rat), monkey or ape. The term "4-1 BB costimulatory domain" refers to amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like. Typical examples of CAR designs are notably provided in Jaspers JE, Brentjens RJ. “Development of CAR T cells designed to improve antitumor efficacy and safety” (Pharmacol Ther. 2017;178:83-91).

In some embodiments, a CAR of the present disclosure further comprises a hinge or spacer region which connects the extracellular antigen binding domain and the transmembrane domain. This hinge or spacer region can be used to achieve different lengths and flexibility of the resulting CAR. Examples of a hinge or spacer region that can be used according to the present disclosure include, but are not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies, or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences, for example peptide sequences, or combinations thereof. Other hinge or spacer regions will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the present disclosure.

In some embodiments, a CAR of the present disclosure further comprises a "linker domain" or "linker region" that connects different domains of the CAR. This domain includes an oligo- or polypeptide region from about 1 to 100 amino acids in length. Suitable linkers will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the present disclosure.

In some embodiments, a CAR of the present disclosure further comprises a "leader sequence".

A CAR of the present disclosure may further include a label, for example a label that facilitates imaging, such as a fluorescent label or other tag. This can, for example be used in methods for imaging tumor binding. The label may be conjugated to the antigen binding domain.

The CARs described herein may be synthesized as single polypeptide chains. In this embodiment, the antigen- specific targeting regions are at the N- terminus, arranged in tandem and are separated by a linker peptide.

Nucleic acids, vectors, host cells

The present disclosure also provides isolated nucleic acids encoding a single domain antibody or a variant thereof, a multivalent binding compound, or a CAR as previously described and nucleic acid constructs comprising thereof. In particular, the present disclosure also encompasses nucleic acid encoding sdAb fused to a Fc portion (typically) a CH2CH3 portion of a human IgG (notably a human IgGl) as previously described.

A nucleic acid according to the present disclosure may be obtained by well-known methods of recombinant DNA technology and/or of chemical DNA synthesis. Also within the scope of the present disclosure, are sequences with at least 60%, 70%, 80% or 90% sequence identity thereto.

The term "nucleic acid," "polynucleotide," or "nucleic acid molecule" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination of a DNA or RNA. RNA includes in vitro transcribed RNA or synthetic RNA or an mRNA sequence encoding a CAR polypeptide as described herein. The nucleic acid may further comprise a suicide gene. The construct may be in the form of a plasmid, vector, transcription or expression cassette.

The present disclosure thus also provides a recombinant expression cassette comprising a nucleic acid according to the present disclosure under the control of a transcriptional promoter allowing the regulation of the transcription of said nucleic acid in a host cell. Said nucleic acid can also be linked to appropriate control sequences allowing the regulation of its translation in a host cell.

The present disclosure also provides a recombinant vector (e.g., a recombinant expression vector) comprising a nucleic acid according to the present disclosure. Advantageously, said recombinant vector is a recombinant expression vector comprising an expression cassette according to the present disclosure.

The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

A vector according to the present disclosure is preferably a vector suitable for stable gene transfer and long-term gene expression into mammalian cells, such as by replication of the sequence of interest, expression of this sequence, maintaining of this sequence in extrachromosomal form, or else integration into the chromosomal material of the host. The recombinant vectors are constructed using standard recombinant DNA technology techniques and produced using conventional methods that are known in the art.

In some embodiments, a vector of the present disclosure is an integrating vector, such as an integrating viral vector, such as in particular a retrovirus or AAV vector. Preferably, the viral vector is a lentiviral vector, most preferably an integrating viral vector.

Within the context of the present disclosure, a "lentiviral vector" means a nonreplicating non-pathogenic virus engineered for the delivery of genetic material into cells, and requiring lentiviral proteins (e.g., Gag, Pol, and/or Env) that are provided in trans. Indeed, the lentiviral vector lacks expression of functional Gag, Pol, and Env proteins. The lentivirus vector is advantageously a self-inactivating vector (SIN vector). The lentiviral vector comprises advantageously a central polypurine tract/DNA FLAP sequence (cPPT-FLAP), and/or insulator sequence (s) such as chicken beta-globin insulator sequence(s) to improve expression of the gene(s) of interest. The lentiviral vector is advantageously pseudotyped with another envelope protein, preferably another viral envelope protein, preferably the vesicular stomatis virus (VSV) glycoprotein. In some preferred embodiments, said lentiviral vector is a human immunodeficiency virus (HIV) vector.

Lentiviral vectors derive from lentiviruses, in particular human immunodeficiency virus (HIV-1 or HIV-2), simian immunodeficiency virus (SIV), equine infectious encephalitis virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV) and feline immunodeficiency virus (FIV), which are modified to remove genetic determinants involved in pathogenicity and introduce new determinants useful for obtaining therapeutic effects.

The lentiviral vector may be present in the form of an RNA or DNA molecule, depending on the stage of production or development of said retroviral vectors. The lentiviral vector can be in the form of a recombinant DNA molecule, such as a plasmid, or in the form of a lentiviral vector particle (interchangeably named lentiviral particle in the context of the present disclosure), such as an RNA molecule(s) within a complex of lentiviral and other proteins.

Such vectors are based on the separation of the cis- and trans-acting sequences. In order to generate replication-defective vectors, the trans-acting sequences (e.g., gag, pol, tat, rev, and env genes) can be deleted and replaced by an expression cassette encoding a transgene. Efficient integration and replication in non-dividing cells generally require the presence of two c/s-acting sequences at the center of the lentiviral genome, the central polypurine tract (cPPT) and the central termination sequence (CTS). These lead to the formation of a triplestranded DNA structure called the central DNA "flap", which acts as a signal for uncoating of the pre-integration complex at the nuclear pore and efficient importation of the expression cassette into the nucleus of non-dividing cells, such as dendritic cells. In one embodiment, the present disclosure encompasses a lentiviral vector comprising a central polypurine tract and central termination sequence referred to as cPPT/CTS sequence as described, in particular, in the European patent application EP 2 169 073.

Further sequences are usually present in cis, such as the long terminal repeats (LTRs) that are involved in integration of the vector proviral DNA sequence into a host cell genome. Vectors may be obtained by mutating the LTR sequences, for instance, in domain U3 of said LTR (AU3) (Miyoshi H et al, 1998, J Virol. 72(10):8150-7; Zufferey et al., 1998, J V/ro/ 72(12):9873-80). Preferably, the vector does not contain an enhancer. In one embodiment, the present disclosure encompasses a lentiviral vector comprising LTR sequences, preferably with a mutated U3 region (AU3) removing promoter and enhancer sequences in the 3' LTR.

The packaging sequence ψ (psi) can also be incorporated to help the encapsidation of the polynucleotide sequence into the vector particles (Kessler et al., 2007, Leukemia, 21 (9): 1859-74; Paschen et al., 2004, Cancer Immunol Immunother 12(6): 196-203). In one embodiment, the present disclosure encompasses a lentiviral vector comprising a lentiviral packaging sequence ψ (psi).

Further additional functional sequences, such as a transport RNA-binding site or primer binding site (PBS) or a Woodchuck PostTranscriptional Regulatory Element (WPRE), can also be advantageously included in the lentiviral vector polynucleotide sequence of the present disclosure, to obtain a more stable expression of the transgene in vivo, can also be advantageously included in the lentiviral vector polynucleotide sequence of the present disclosure, to obtain a more stable expression of the transgene in vivo. In some embodiments, the present disclosure encompasses a lentiviral vector comprising a PBS. In some embodiments, the present disclosure encompasses a lentiviral vector comprising a WPRE and/or an IRES.

Thus, in a preferred embodiment, the lentiviral vector comprises at least one cPPT/CTS sequence, one ψ sequence, one (preferably 2) LTR sequence, and an expression cassette including a transgene under the transcriptional control of a β2ηη or class I MHC promoter. In some embodiments of the present disclosure, a vector (i.e. a recombinant transfer vector) of the present disclosure is an expression vector comprising appropriate means for expression of the hook fusion protein and/or the target fusion protein in a host cell.

Various promoters may be used to drive high expression of the nucleic acid sequence encoding the hook fusion protein and/or the target fusion protein. The promoter may be a tissuespecific, ubiquitous, constitutive or inducible promoter. Preferred promoters are notably functional in T cells and/or NK cells, preferably human T cells and human NK cells. In particular, preferred promoters are able to drive high expression of the hook fusion protein and the target fusion protein (notably a CAR as previously defined) from lentivectors in T cells or NK cells, preferably human T cells or NK T cells. For example, a promoter according to the present disclosure can be selected from phosphoglycerate kinase promoter (PGK), spleen focusforming virus (SFFV) promoters, elongation factor- 1 alpha (EF-1 alpha) promoter including the short form of said promoter (EFS), viral promoters such as cytomegalovirus (CMV) immediate early enhancer and promoter, retroviral 5’ and 3’ LTR promoters including hybrid LTR promoters, human ubiquitin promoter, MHC class I promoter, MHC class II promoter, and β2 microglobulin ( β2ηη) promoter. The promoters are advantageously human promoters, i.e., promoters from human cells or human viruses such as spleen focus-forming virus (SFFV). Human ubiquitin promoter, MHC class I promoter, MHC class II promoter, and β2 microglobulin ( β2ηη) promoter are more particular preferred. Preferably, the MHC class I promoter is an HLA-A2 promoter, an HLA-B7 promoter, an HLA-Cw5 promoter, an HLA-F, or an HLA-E promoter. In some embodiments the promoter is not a CMV promoter/enhancer, or is not a dectin-2 or MHCII promoter. Such promoters are well-known in the art and their sequences are available in sequence data base.

Typically, lentiviral particles refer to the extracellular infectious form of a virus composed of genetic material made from either DNA or RNA (most preferably single stranded RNA) surrounded by a protein coat, called the capsid, and in some cases an envelope of lipids that surrounds the capsid. Thus, a lentiviral vector particle (or a lentiviral particle) comprises a lentiviral vector as previously defined in association with viral proteins. The vector is preferably an integrating vector.

The RNA sequences of the lentiviral particle can be obtained by transcription from a double- stranded DNA sequence inserted into a host cell genome (proviral vector DNA) or can be obtained from the transient expression of plasmid DNA (plasmid vector DNA) in a transformed host cell. Appropriate methods for designing and preparing lentiviral particles in particular for therapeutic application are well-known in the art and are for example described in Merten OW, Hebben M, Bovolenta C. Production of lentiviral vectors. Mol Ther Methods Clin Dev. 2016 Apr 13;3: 16017.

Preferably the lentiviral particles have the capacity for integration. As such, they contain a functional integrase protein. Non-integrating vector particles have one or more mutations that eliminate most or all of the integrating capacity of the lentiviral vector particles. For, example, a non-integrating vector particle can contain mutation(s) in the integrase encoded by the lentiviral pol gene that cause a reduction in integrating capacity. In contrast, an integrating vector particle comprises a functional integrase protein that does not contain any mutations that eliminate most, or all of the integrating capacity of the lentiviral vector particles.

In some embodiments, the present disclosure encompasses a vector system comprising one or more vector comprising:

(a) a nucleic acid comprising a nucleic acid sequence encoding a chimeric antigen receptor as previously defined, and optionally

(b) a nucleic acid encoding another protein or polypeptide wherein the nucleic acids (a) and (b) are located on the same or on separated vectors.

Preferred nucleic acids (a) have been described in the prior section.

When the vector system comprises more than one vector, typically two or more vectors, said vectors are typically of the same type (e.g.: a lentiviral vector). In the following sections the vector can also be intended as “the one or more vector” or “the vector system”. Preferably the present disclosure encompasses a lentiviral vector system and notably a lentiviral particle system.

According to the present disclosure, the vector can be an expression vector. The vector can be a plasmid vector.

In one embodiment of the present disclosure, the nucleic acid encoding the CAR and the other protein are inserted into separate vectors.

In another embodiment, the nucleic acid encoding the CAR and the other protein are inserted into the same vector.

In the later embodiment, each coding sequence (i.e. the nucleic acids encoding respectively the other protein or polypeptide and the CAR) can be inserted in a separate expression cassette. Each expression cassette therefore comprises the coding sequence (open reading frame or ORF) functionally linked to the regulatory sequences which allow the expression of the corresponding protein in the host cell, such as in particular promoter, promoter/enhancer, initiation codon (ATG), codon stop, transcription termination signal.

Alternatively, the proteins may also be expressed from a unique expression cassette using an Internal Ribosome Entry Site (IRES), or a self-cleaving 2A peptide inserted between the two coding sequences to allow simultaneous expression.

Nucleic acids encoding the proteins can be inserted in a single expression vector, said single vector comprising a bicistronic expression cassette. Vectors containing biscitronic expression cassette are well known in the art. Advantageously, bicistronic expression cassettes contain an Internal Ribosome Entry Site (IRES) that enables the expression of both fusion proteins from a single promoter. Suitable commercially available bicistronic vectors can include, but are not limited to plasmids of the pIRES (Clontech), pBud (Invitrogen) and Vitality (Stratagene) series. Preferably, the nucleic acid located upstream of the IRES sequence is operably-linked to a promoter. Preferably the nucleic acid encoding the hook protein is inserted upstream of the IRES sequence and the nucleic acid encoding the target fusion protein is inserted downstream of said IRES sequence to ensure that enough the hook fusion protein will be sufficiently expressed to retain every target fusion protein. In some embodiments multicistronic expression vectors may be used wherein more than one, typically at least two, nucleic acids encoding each a distinct hook and at least one nucleic acid encoding a target fusion protein are inserted.

A self-cleaving 2A peptide can also be used in replacement of IRES. Such strategy is highly advantageous because of its small size and high cleavage and translation efficacy between nucleic acid sequences upstream and downstream of the 2A peptide. Suitable 2A peptide according to the present disclosure are notably described in Kim JH, Lee S-R, Li L-H, et al. High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice. PLoS ONE. 2011;6(4):el8556, but see also Liu, Z., O. Chen, LB. J. Wall, M. Zheng, Y. Zhou, L. Wang, H. Ruth Vaseghi, L. Qian, and J. Liu. 2017. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Scientific reports. 7:2193.. 2A peptides can be selected from FMDV 2A (abbreviated herein as F2A); equine rhinitis A virus (ERAV) 2A (E2A); porcine teschovirus-1 2A (P2A) and Thoseaasigna virus 2A (T2A). P2A or T2A peptide is preferred. The present disclosure also encompasses a viral particle system, wherein the one or more viral particle comprises a viral vector, typically an integrating viral vector, as previously defined. Preferably, the viral vector is a lentiviral vector and the viral particle is a lentiviral particle. In one embodiment, the viral particle system comprises separated particles comprising a viral vector encoding respectively the hook protein and the CAR. In an alternative embodiment, the viral particle system comprises one particle comprising viral vector encoding both the hook fusion protein and the CAR as previously described. The nucleic acid sequence encoding the hook protein and the nucleic acid sequence encoding the CAR are preferably expressed from a unique expression cassette as defined above.

The present disclosure also provides a host cell containing a nucleic acid construct as herein disclosed, notably a recombinant expression cassette or a recombinant vector according to the present disclosure. The host cell is either a prokaryotic or eukaryotic host cell. The terms "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

The present disclosure also provides a method for producing in a host cell as defined above a polypeptide, consisting or comprising a single domain antibody or a CAR as previously defined, comprising the steps of: providing a host cell containing a nucleic acid construct, a recombinant expression cassette or a recombinant vector according to the present disclosure, culturing said host cell, and optionally purifying the single domain antibody or CAR of the present disclosure.

Methods for purifying polypeptides are well known in the art, such as chromatography (e.g., ion exchange chromatography, gel permeation chromatography and reversed phase chromatography ) .

The present disclosure also encompasses compositions comprising a nucleic acid construct as herein disclosed.

Immune cells and method for obtaining thereof The present disclosure also provides isolated cells, populations of cells, cell lines, or cell cultures, comprising a nucleic acid construct as previously described, notably vectors and more particularly a viral vector particle encoding at least one or more CAR as previously described. Preferably the vectors and /or lentiviral particles further comprise a nucleic acid sequence encoding a hook protein.

In one embodiment, the cell contains the vector and/or viral vector particle integrated into the cellular genome. In one embodiment, the cell contains the vector stably expressing the CAR. In one embodiment, the cell produces lentiviral vector particles encoding the CARs.

The cells are preferably mammalian cells, particularly human cells. Particularly preferred are human non-dividing cells. Preferably, the cells are immune cells, As used herein, the term “immune cells” includes cells that are of hematopoietic origin and that play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells, natural killer cells (NK cells), myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

As used herein, the term “T cell” includes cells bearing a T cell receptor (TCR), T-cells according to the present disclosure can be selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes, Mucosal-Associated Invariant T cells (MAIT), Yδ T cell, tumour infiltrating lymphocyte (TILs) or helper T- lymphocytes included both type 1 and 2 helper T cells and Th 17 helper cells. In another embodiment, said cell can be derived from the group consisting of CD4+ T- lymphocytes and CD8+ T-lymphocytes.

Said immune cells may originate from a healthy donor or from a subject suffering from a cancer.

Immune cells can be extracted from blood or derived from stem cells. The stem cells can be adult stem cells, embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human cells are CD34+ cells.

T-cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T-cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as FICOLL™ separation. In one embodiment, cells from the circulating blood of a subject are obtained by apheresis. In certain embodiments, T-cells are isolated from PBMCs. PBMCs may be isolated from buffy coats obtained by density gradient centrifugation of whole blood, for instance centrifugation through a LYMPHOPREP™ gradient, a PERCOLL™ gradient or a FICOLL™ gradient. T-cells may be isolated from PBMCs by depletion of the monocytes, for instance by using CD 14 DYNABEADS®. In some embodiments, red blood cells may be lysed prior to the density gradient centrifugation.

In another embodiment, said cell can be derived from a healthy donor, from a subject diagnosed with cancer, notably with Ewing sarcoma. The cell can be autologous or allogeneic.

In allogeneic immune cell therapy, immune cells are collected from healthy donors, rather than the patient. Typically, these are HLA matched to reduce the likelihood of graft vs. host disease. Alternatively, universal ‘off the shelf’ products that may not require HLA matching comprise modifications designed to reduce graft vs. host disease, such as disruption or removal of the TCRαβ receptor. See Graham et al., Cells. 2018 Oct; 7(10): 155 for a review. Because a single gene encodes the alpha chain (TRAC) rather than the two genes encoding the beta chain, the TRAC locus is a typical target for removing or disrupting TCRαβ receptor expression. Alternatively, inhibitors of TCRαβ signalling may be expressed, e.g. truncated forms of CD3ζ can act as a TCR inhibitory molecule. Disruption or removal of HLA class I molecules has also been employed. Typically, gene disruption may be achieved using gene editing techniques such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases can advantageously used (see Li, H., Yang, Y, Hong, W. et al. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Sig Transduct Target Ther 5, 1 (2020)). For example, Torikai et al., Blood. 2013; 122: 1341— 1349 used ZFNs to knock out the HLA- A locus, while Ren et al., Clin. Cancer Res. 2017;23:2255-2266 knocked out Beta-2 microglobulin (B2M), which is required for HLA class I expression. Ren et al. simultaneously knocked out TCRαβ, B2M and the immune-checkpoint PD1.

Generally, the immune cells are activated and expanded to be utilized in the adoptive cell therapy. The immune cells as herein disclosed can be expanded in vivo or ex vivo. The immune cells, in particular T-cells can be activated and expanded generally using methods known in the art. Generally, the T-cells are expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells.

Typically, the immune cell is modified to express chimeric antigen receptor as herein disclosed. Expression of multiple tumor- specific targets may reduce the chance of antigen escape by mutating or reducing expression of the target antigen. As previously described the CARs of the present disclosure may be multispecific CARs (i.e. directed against more than one antigen, that is directed against GPC4 and at least another antigen). In addition, or alternatively, an immune cell as herein described may express one or more CAR(s) as herein defined and at least another CAR targeting one or more nother antigen(s).

Methods by which immune cells can be genetically modified by repressing the expression of specific molecules and/or to express a recombinant antigen receptor are well known in the art. A nucleic acid molecule encoding the antigen receptor may be introduced into the cell in the form of e.g. a vector (such as viral or nonviral DNA plasmid-based vectors) or any other suitable nucleic acid construct. Typically, in some embodiments non-viral vectors strategies can be preferred to avoid major disadvantages of viral-based delivery systems. For example, recombinant expression may be achieved using transposon based expression such as typically the Sleeping Beauty (SB) transposon system (see Molecular reconstruction of Sleeping Beauty, a Tcl-like transposon from fish, and its transposition in human cells. Ivies Z, Hackett PB, Plasterk RH, Izsvak Z Cell. 1997 Nov 14; 91 (4):501 - 10 or for review Hackett PB, Largaespada DA, Cooper LJ. A transposon and transposase system for human application. Mol Ther. 2010;18(4):674-683; and Aronovich EL, Mclvor RS, Hackett PB. The Sleeping Beauty transposon system: a non-viral vector for gene therapy. Hum Mol Genet. 2011;20(Rl):R14- R20. ) or PiggyBac transposon system (see Woodard LE, Wilson MH. piggyBac-ing models and new therapeutic strategies. Trends Biotechnol. 2015;33(9):525-533; Ivies Z, Li MA, Mates L, et al. Transposon-mediated genome manipulation in vertebrates. Nat Methods. 2009;6(6):415-422; Li X, Bumight ER, Cooney AL, et al. piggyBac transposase tools for genome engineering. Proc Natl Acad Sci U S A. 2013;110(25):E2279-E2287; and Zhao, Shuang et al. “PiggyBac transposon vectors: the tools of the human gene encoding.” Translational lung cancer research vol. 5,1 (2016): 120-5)). Typically also, genome editing techniques such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases can advantageously used (Li, H., Yang, Y, Hong, W. et al. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Sig Transduct Target Ther 5, 1 (2020); but see for example regarding the CRISPR-Cas system the recent works of Miura, H., Quadros, R., Gurumurthy, C. et al. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc 13, 195-215 (2018); Hendel, A., Bak, R., Clark, J. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol 33, 985-989 (2015); Roth, T.L., Puig-Saus, C., Yu, R. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405-409 (2018). https://doi.org/10.1038/s41586- 018-0326-5 or Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113-117 (2017)).

Vectors, and their required components, are well known in the art. Nucleic acid molecules encoding antigen receptors can be generated using any method known in the art, e.g. molecular cloning using PCR. Antigen receptor sequences can be modified using commonly used methods, such as site-directed mutagenesis.

In another aspect, the present disclosure relates to an ex vivo method for generating a population of cells for use in adaptive immunotherapy comprising transforming said cell with a CAR as herein described.

Compositions and kits of the present disclosure

The present disclosure also encompasses pharmaceutical compositions comprising one or more anti-GPC4 single domain antibody(ies), CAR(s), nucleic acid construct encoding thereof and/or one or more isolated cell(s) or cell population(s) comprising a CAR as herein disclosed, alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier and optionally formulated with formulated with sterile pharmaceutically acceptable buffer(s), diluent(s), and/or excipient(s). Pharmaceutically acceptable carriers typically enhance or stabilize the composition, and/or can be used to facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, and in some embodiments, pharmaceutically inert.

Administration of a pharmaceutical composition comprising sdAbs as herein disclosed can be accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial (directly to the tumor), intramuscular, spinal, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.

The genetically modified cells or pharmaceutical composition of the present disclosure can be administered by any convenient route, including parenteral administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Compositions can take the form of one or more dosage units.

Thus, in addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.).

Depending on the route of administration, the single domain antibody or variant thereof, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The composition is typically sterile and preferably fluid. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum mono stearate or gelatin.

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl, cellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, ie. dosage.

Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical compositions of the present disclosure may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Pharmaceutical compositions of the disclosure can be prepared in accordance with methods well known and routinely practiced in the art. See. e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Pharmaceutical compositions are preferably manufactured under GMP conditions.

The amount of the pharmaceutical composition of the present disclosure that is effective/active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.

The compositions as herein disclosed comprise an effective amount of a binding molecule of the present disclosure (e.g. a single domain antibody or variant thereof or a chimeric antigen receptor) such that a suitable dosage will be obtained. The correct dosage of the compounds will vary according to the particular formulation, the mode of application, and its particular site, host and the disease being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated dose.

Typically, this amount is at least about 0.01 % of a binding molecule of the present disclosure by weight of the composition. Preferred compositions of the present disclosure are prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the binding molecule of the present disclosure.

For intravenous administration, the composition can comprise from about typically about 0.1 mg/kg to about 250 mg/kg of the animal's body weight, preferably, between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, and more preferably about 1 mg/kg to about 10 mg/kg of the animal's body weight.

The present compositions can take the form of suitable carriers, such aerosols, sprays, suspensions, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

The pharmaceutical compositions as herein disclosed can be co-administered with other therapeutics, for example anti-cancer agents. Medical uses

The present disclosure also relates to an anti-GPC4 single domain antibody or variant thereof as herein described, a CAR directed against GPC4 or variant thereof as herein described, a nucleic acid encoding said anti-GPC4 single domain antibody or CAR, or to a cell, line or cell population comprising a CAR as described herein for use in the treatment of a disease. The present disclosure also relates to an anti-GPC4 single domain antibody or variant thereof as herein described, a CAR directed against GPC4 or variant thereof as herein described, a nucleic acid encoding said anti-GPC4 single domain antibody or CAR, or to a cell, line or cell population comprising said CAR as described herein in the manufacture of a medicament.

In more specific embodiments, the diseases include proliferative diseases, neurodegenerative diseases and metabolic disorders. Typically, the disease, in particular the proliferative disease, or the metabolic disorder, is a GPC4-associated disease, typically a disease which is associated with an increased expression of GPC4, and/or wherein reduced expression of GPC4, or GPC4 knock-down, is associated with an improvement of the disease (see notably references 16-19)

Proliferative disorders are diseases associated with abnormal and/or uncontrolled cell proliferation and comprise cancer, atherosclerosis, rheumatoid arthritis, psoriasis, idiopathic pulmonary fibrosis, scleroderma and cirrhosis of the liver. Typically, the proliferative disease is cancer.

Metabolic disorders include but is not limited to prediabetes, diabetes (type I and/or type II), metabolic syndrome, obesity, high body fat content, insulin resistance, and nonalcoholic fatty liver disease. Metabolic syndrome is a clustering of at least three of the five following medical conditions: abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, and low serum high-density lipoprotein (HDL).

Neurodegenerative diseases typically include Parkinson disease and Alzheimer disease.

The cancer can be a solid cancer or a liquid tumor. Cancers that may treated by methods, uses and compositions described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malign melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. More specific cancers which can be treated and/or prevented according to the present disclosure include GPC4-associated cancers. Typically, GPC4-associated cancers are cancers wherein GPC4 is expressed or overexpressed. Typical cancers wherein GPC4 is expressed and/or overexpressed include breast cancers, gastric or stomach cancers, salivary duct carcinoma, lung adenocarcinomas (such as non-small cell lung (NSCLC)), liver cancers, testis cancer, urothelial cancers, prostate cancer, renal cancer, carcinoids, thyroid cancer, skin cancer, ovarian cancers, uterine cancers (such as uterine serous endometrial carcinoma), endometrial cancer, colon cancers, notably colorectal cancers, neuroblastoma, hepatocellular carcinomas, glioblastoma and pancreatic cancers. Typically, the cancer is a breast cancer, a renal cancer, a thyroid cancer, a glioma, a liver cancer, a pancreatic cancer, a carcinoid, a testis cancer, an urothelial cancer, or a skin cancer. More particularly the cancer is a pancreatic cancer or a colorectal cancer.

More specific cancers which can be treated and/or prevented according to the present disclosure include GPC4-associated cancers. Typically, GPC4-associated cancers are cancers wherein GPC4 is expressed or overexpressed. Typical cancers wherein GPC4 is expressed and/or overexpressed include breast cancers, gastric or stomach cancers, salivary duct carcinoma, lung adenocarcinomas (such as non-small cell lung (NSCLC)), liver cancers, testis cancer, urothelial cancers, prostate cancer, renal cancer, carcinoids, thyroid cancer, skin cancer, ovarian cancers, uterine cancers (such as uterine serous endometrial carcinoma), endometrial cancer, colon cancers, notably colorectal cancers, glioblastoma and pancreatic cancers. Typically, the cancer is a breast cancer, a renal cancer, a thyroid cancer, a glioma, a liver cancer, a pancreatic cancer, a carcinoid, a testis cancer, an urothelial cancer, or a skin cancer. More particularly the cancer is a pancreatic cancer or a colorectal cancer. Thanks to its neutralizing capacities a GPC4 sdAb as herein disclosed can also be used to down regulate GPC4 signaling pathway. In particular, an anti-GPC4 sdAb as herein disclosed can be used to self-renewal and differentiation of stem cells (SCs) (13).

Anti-GPC4 sdAbs as herein disclosed may be particularly to promote differentiation of PSPCs toward midbrain dopaminergic (mDA) neurons. It has been shown notably that when transplanted in the brain of a rat models for Parkinson’s disease (PD) these GPC4 mutant PSCs generate greater mDA neuron numbers capable to rescue the motor defects characterizing PD rat models (15). Importantly, these behavioral improvements occur without causing tumor side effects observed using control cells (15). Thus, the biological state conferred to PSCs by GPC4 down-regulation supports efficient and safe production of mDA neurons with high potential clinical relevance for neurodegenerative disease therapy (15).

An anti-GPC4 single domain antibody or variant thereof as herein described, a CAR directed against GPC4 or variant thereof as herein described, a nucleic acid encoding said anti- GPC4 single domain antibody or CAR, or to a cell, line or cell population comprising a CAR as described herein can also be used for cell-based replacement therapy, notably for the treatment of degenerative diseases, notably associated with the loss of mDA neurons such as the Parkinson disease or the Alzheimer disease.

For example, anti-GPC4 sdAbs or their variants thereof as herein described can be used for controlling the generation of midbrain DA neurons in vitro and/or in vivo, notably to increase the efficiency and safety of cell transplantation in neurodegenerative disease. Indeed, anti-GPC4 sdAbs or their variants as herein described can be used to control GPC4 signaling pathway and to reduce Embryonic stem (ES) cells, notably pluripotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs), (differentiated toward the midbrain dopaminergic fate in vitro) tumorigenic features while at the same time increasing neuronal features (see Fico A, de Chevigny A, Melon C, et al. Reducing glypican-4 in ES cells improves recovery in a rat model of Parkinson's disease by increasing the production of dopaminergic neurons and decreasing teratoma formation. J Neurosci. 2014;34(24):8318-8323).

In some embodiments, the present disclosure encompasses a method for producing mDA neurons comprising a step of culturing stems cells, (SCs) such as embryonic stem cells, pluripotent stems cells (PSCs), or induced-pluripotent stem cells (iPSCs) in the presence of an anti-sdAb or a variant thereof, as herein disclosed. Said mDA neurons may be used in cell replacement therapy. Thus, the obtained differentiated mDA neurons may be then transplanted in the brain of patient suffering from a neurodegenerative disease associated with mDA neurons loss such as Parkinson disease or Alzheimer disease. Thus, in some embodiments, the present application encompasses a method of treatment of a neurodegenerative disease associated with a loss of DA neurons, comprising culturing SCs, as above defined, in the presence of anti-GPC4 sdAb(s) of the present disclosure to produce mDA neurons and implanting said mDA neurons in the brain a patient suffering from a neurogenerative disease.

The present disclosure also encompasses a method to modulate the stem cell (SC), notably pluripotent stem cells (PSCs), such as human induced PSCs (hiPSCs) functions in vitro or ex vivo, comprising the culture of stem cells in the presence of an anti-GPC4 sdAb or a variant thereof as herein described. In particular, said anti-GPC4 sdAb or variant thereof can be used to manipulate self-renewal or pluripotency, improve efficiency of lineage entry in differentiation conditions (mesoderm, ectoderm or endoderm), and/or reducing or suppressing tumorigenicity of stem cells for xenografts application. Indeed, downregulation of GPC4 with anti-GPC4 sdAbs stem cells lose self-renewal and undergo improved lineage entry (see also Fico A, De Chevigny A, Egea J, et al. “Modulating Glypican4 suppresses tumorigenicity of embryonic stem cells while preserving self-renewal and pluripotency”. Stem Cells. 2012;30(9): 1863- 1874).

The present disclosure also encompasses a method for the prevention and/or treatment of a proliferative disease, in particular a GPC4-associated proliferative disease as above described or the cell as well as a method for cell therapy, comprising administering to a subject to an anti-GPC4 single domain antibody or variant thereof as herein described, a CAR directed against GPC4 or variant thereof as herein described, a nucleic acid encoding said anti-GPC4 single domain antibody or CAR, or a cell, line or to a cell population comprising a CAR as described herein, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an anti-GPC4 single domain antibody or variant thereof, a CAR, a cell, line or cell population comprising a CAR as described herein and/or of a pharmaceutical composition of the present disclosure. The method may additionally comprise the step of identifying a subject who has a proliferative disease.

The present disclosure also include the use of one or more of the anti-GPC4 single domain antibodies or variants thereof, CARs directed against GPC4 or variants thereof , nucleic acids encoding said anti-GPC4 single domain antibodies or CARs, cell lines or cell population comprising a CAR as described herein in targeted immune therapy. For example sdAbs of the present disclosure and in particular variants thereof in the form of multispecific polypeptides further targeting an immune cell antigen, and CAR expressing immune cells (notably CAR T cells) may be used in immune cell redirecting immune therapies.

In another aspect, the present disclosure relates to a method for stimulating a T cell- mediated immune response to a target cell population or tissue in a subject, the method comprising administering to a subject an effective amount of a cell or cell population that expresses a CAR directed against GPC4 as herein described.

In another aspect, the present disclosure relates to a method of providing an anti-tumor immunity in a subject, the method comprising administering to the mammal an effective amount of a cell or cell population genetically modified to express a CAR directed against GPC4 as herein described, thereby providing an anti- tumor immunity in the subject.

The present disclosure also relates to an anti-GPC4 single domain antibody (including variants thereof), a CAR directed against GPC4 as herein described, or a nucleic acid construct encoding said anti-GPC4 SdAb or CAR, or to an immune cell expressing said CAR, as previously defined, for use in adoptive cell or CAR-T cell therapy in a subject. Typically, the immune cell for use in the method of the present disclosure is a redirected T-cell, e.g. a redirected CD8+ and/ or CD4+ T-cell.

In some embodiments, anti-GPC4 single domain antibodies (including variants thereof), and CARs directed against GPC4 as herein described, as well as nucleic acid constructs encoding them and cells comprising such CARs are useful for inhibiting tumor growth, inducing differentiation, reducing tumor volume, and/or reducing the tumorigenicity of a tumor. The methods of use can be in vitro, ex vivo, or in vivo methods.

In certain aspects, the subject is a human. In certain aspects, the subject has a tumor or has had a tumor removed. The subject can also be at risk of developing a cancer. In some embodiments, the subject is receiving other cancer therapeutics, such as immune therapy, chemotherapy, hormone therapy or radiotherapy.

In some embodiments, cancer treatment, and/or adoptive cell cancer therapy as above described are administered in combination with additional cancer therapies. , In some embodiments, cancer treatment and/or adoptive cell cancer therapy as above described are administered in combination with targeted therapy, immunotherapy such as immune checkpoint therapy and immune checkpoint inhibitor, co- stimulatory antibodies, chemotherapy, hormone therapy and/or radiotherapy. For example for patients suffering from a pancreatic cancer, cancer therapy as herein described could be administered in combination with chemotherapy.

Immune checkpoint therapy such as checkpoint inhibitors include, but are not limited to programmed death-1 (PD-1) inhibitors, programmed death ligand-1 (PD-L1) inhibitors, programmed death ligand-2 (PD-L2) inhibitors, lymphocyte-activation gene 3 (LAG3) inhibitors, T-cell immunoglobulin and mucin-domain containing protein 3 (TIM-3) inhibitors, T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitors, B- and T-lymphocyte attenuator (BTLA) inhibitors, V-domain Ig suppressor of T-cell activation (VISTA) inhibitors, cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors, Indoleamine 2,3- dioxygenase (IDO) inhibitors, killer immunoglobulin-like receptors (KIR) inhibitors, KIR2L3 inhibitors, KIR3DL2 inhibitors and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) inhibitors. In particular, checkpoint inhibitors include antibodies anti-PDl, anti- PD-L1, anti-CTLA-4, anti-TIM-3, anti-LAG3. Co-stimulatory antibodies deliver positive signals through immune-regulatory receptors including but not limited to ICOS, CD137, CD27, OX-40 and GITR.

Example of anti-PDl antibodies include, but are not limited to, nivolumab, cemiplimab (REGN2810 orREGN-2810), tislelizumab (BGB-A317), tislelizumab, spartalizumab (PDR001 or PDR-001), ABBV-181, JNJ-63723283, BI 754091, MAG012, TSR-042, AGEN2034, pidilizumab, nivolumab (ONO-4538, BMS-936558, MDX1106, GTPL7335 or Opdivo), pembrolizumab (MK-3475, MK03475, lambrolizumab, SCH-900475 or Keytruda) and antibodies described in International patent applications W02004004771, W02004056875, W02006121168, WO2008156712, W02009014708, W02009114335, WO2013043569 and W02014047350.

Example of anti-PD-Ll antibodies include, but are not limited to, LY3300054, atezolizumab, durvalumab and avelumab.

Example of anti-CTLA-4 antibodies include, but are not limited to, ipilimumab (see, e.g., US patents US6,984,720 and US8,017,114), tremelimumab (see, e.g., US patents US7, 109,003 and US8, 143,379), single chain anti-CTLA4 antibodies (see, e.g., International patent applications WO1997020574 and WO2007123737) and antibodies described in US patent US8,491,895.

Example of anti-VISTA antibodies are described in US patent application US20130177557. Example of inhibitors of the LAG3 receptor are described in US patent US5,773,578.

Example of KIR inhibitor is IPH4102 targeting KIR3DL2.

As used herein, the term “chemotherapy” has its general meaning in the art and refers to the treatment that consists in administering to the patient a chemotherapeutic agent. A chemotherapeutic entity as used herein refers to an entity which is destructive to a cell, that is the entity reduces the viability of the cell. The chemotherapeutic entity may be a cytotoxic drug. Chemotherapeutic agents include, but are not limited to alkylating agents such as thiotepa and cyclo sphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimu stine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5- FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; ellip tinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; anthracyclines, nitrosoureas, antimetabolites, epipodophy lotoxins, enzymes such as L-asparaginase; anthracenediones; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide; biological response modifiers such as IFNa, IL-2, G-CSF and GM-CSF; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Suitable examples of radiation therapies include, but are not limited to external beam radiotherapy (such as superficial X-rays therapy, orthovoltage X-rays therapy, megavoltage X- rays therapy, radiosurgery, stereotactic radiation therapy, Fractionated stereotactic radiation therapy, cobalt therapy, electron therapy, fast neutron therapy, neutron-capture therapy, proton therapy, intensity modulated radiation therapy (IMRT), 3-dimensional conformal radiation therapy (3D-CRT) and the like); brachytherapy; unsealed source radiotherapy; tomotherapy; and the like. Gamma rays are another form of photons used in radiotherapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, radiotherapy may be proton radiotherapy or proton minibeam radiation therapy. Proton radiotherapy is an ultra-precise form of radiotherapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R. Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy. Int J Radiat Oncol Biol Phys. 2019 Jun l;104(2):266- 271. doi: 10.1016/j.ijrobp.2019.01.080; Prezado Y, Jouvion G, Patriarca A, Nauraye C, Guardiola C, Juchaux M, Lamirault C, Labiod D, Jourdain L, Sebrie C, Dendale R, Gonzalez W, Pouzoulet F. Proton minibeam radiation therapy widens the therapeutic index for high-grade gliomas. Sci Rep. 2018 Nov 7;8(1):16479. doi: 10.1038/s41598-018-34796-8). Radiotherapy may also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation. FLASH radiotherapy involves the ultra-fast delivery of radiation treatment at dose rates several orders of magnitude greater than those currently in routine clinical practice (ultra-high dose rate) (Favaudon V, Fouillade C, Vozenin MC. The radiotherapy FLASH to save healthy tissues. Med Sci (Paris) 2015 ; 31 : 121-123. DOI: 10.105 l/medsci/20153102002); Patriarca A., Fouillade C. M., Martin F., Pouzoulet F., Nauraye C., et al. Experimental set-up for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), pp. 619-626. doi: 10.1016/j.ijrobp.2018.06.403. Epub 2018 Jul 11).

“In combination” may refer to administration of the additional therapy before, at the same time as or after administration of the T cell composition according to the present disclosure.

In addition, or as an alternative to the combination with checkpoint blockade, the T cell composition of the present disclosure may also be genetically modified to render them resistant to immune-checkpoints using gene-editing technologies including but not limited to TALEN and Crispr/Cas. Such methods are known in the art, see e.g. US20140120622. Gene editing technologies may be used to prevent the expression of immune checkpoints expressed by T cells (see the above listed checkpoint inhibitors) and more particularly but not limited to PD-1, Lag-3, Tim-3, TIGIT, BTLA CTLA-4 and combinations of these. The T cell as discussed here may be modified by any of these methods. The T cell according to the present disclosure may also be genetically modified to express molecules increasing homing into tumors and or to deliver inflammatory mediators into the tumor microenvironment, including but not limited to cytokines, soluble immune-regulatory receptors and/or ligands.

Having thus described different embodiments of the present disclosure, it should be noted by those skilled in the art that the disclosures herein are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present disclosure. Accordingly, the present disclosure is not limited to the specific embodiments as illustrated herein.

Diagnostic tool

Nanobodies can aid in early diagnosis and cancer prevention by detecting or defining biomarkers. Nanobodies can improve current mAb-based diagnostic techniques due to their high specificity. Furthermore, their high stability under extremes of temperature, pH, or ionic strength, ensures that the application still can occur under harsh conditions.

Typically, anti-GPC4 sdAb as per the present disclosure can be used in cell-based ELISA assays. To perform sandwich ELISA, both a capturing and detecting nanobody are used, preferably targeting different epitopes on the antigen.

The small size of nanobodies is highly advantageous especially in the field of molecular imaging as it enables rapid tumor accumulation and homogenous distribution as well as efficient blood clearance, contributing to high tumor-to-background ratios. Moreover, nanobodies can be easily conjugated to several kinds of imaging agents and their high specificity renders their use relatively safe. Single-photon emission computed tomography (SPECT) is based on γ-rays and sdAb of the present disclosure can thus linked to radionuclides such as ^99mTc, ¹⁷⁷Lu, ¹²³I and ¹¹¹ln. On the other hand, the positron-emitting radioisotopes ⁶⁸Ga, ¹²⁴I or ⁸⁹Zr can be used for positron emission tomography (PET) purposes.

In some embodiments of the present disclosure, the anti-GPC4 single domain antibodies as herein described are useful for detecting the presence of GPC4 in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. As used herein, the term “biological sample” is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a patient, as well as tissues, cells and fluids present within a patient, or subject. In certain aspects, a biological sample comprises one or more cell(s) or tissue(s). In some embodiments, the biological sample can be a fixed tissue sample. In certain aspects, such tissues include normal and/or cancerous tissues that express GPC4, notably that express GPC4 at higher levels relative to other tissues or similar tissue from a control subject or from a control population of subjects.

Also included is an in vitro method of diagnosing a GPC4-associated disease or disorder, typically a GPC4-associated cancer or tumor, notably a cancer, or a metabolic disease, as above defined. In certain aspects, the method comprises: contacting a (tested) biological sample obtained from a subject with an anti-GPC4 single domain antibody of the present disclosure; determining the level of expression (either quantitatively or qualitatively) of GPC4 in said sample by detecting the binding of said anti-GPC4 sdAb to GPC4 expressed by the sample; and comparing the level of expression of GPC4 in said sample with a reference value; wherein a higher level of expression of GPC4 in the biological sample as compared reference value indicates the presence of a GPC4-associated disease. In certain aspects, biological sample is obtained from an individual suspected of or having a GPC4 associated disease. In certain aspects, the disease is a metabolic disorder, or a cell proliferative disorder, such as a cancer or a tumor as previously defined.

Typically, the reference value can be the level of GPC4 expression in the corresponding control tissue, and in particular in the corresponding control cell. In some embodiments, the reference value can be obtained from a control, or reference, sample. The control sample can be a sample from the corresponding normal tissue obtained from the same subject or patient as the tested sample, from a control healthy subject or from a control population of healthy subjects.

In certain aspects, a method of diagnosis or detection, such as those described above, comprises detecting binding of an anti-GPC4 single domain antibody expressed on the surface of one or more cells or in a membrane preparation obtained from one or more cells expressing GPC4 on their surface. An exemplary assay for detecting binding of an anti-GPC4 sdAb to GPC4 expressed on the surface of a cell is a "FACS" assay. In one embodiment, an anti-GPC4 sdAb is used to select subjects eligible for therapy with an anti-GPC4 treatment or therapy, typically, wherein GPC4 is a biomarker for the selection of patients. The disclosure further provides for the use of an anti-GPC4 sdAb in a method of diagnosing a subject suffering from a disorder associated with an increased GPC4 expression (e.g., a cancer), the method comprising: determining the presence or expression level of GPC4 in a sample obtained from the subject by contacting the sample with an anti- GCP4 sdAb as herein described and detecting the presence of the bound sdAb. The anti-GPC4 therapy is typically an anti-GPC4 antibody or a variant thereof, typically an anti-GPC4 sdAb as herein disclosed or a variant thereof, a multivalent binding compound or a chimeric antigen receptor as also disclosed herein.

Certain other methods can be used to detect binding of anti-GPC4 sdAb, as herein disclosed, to GPC4. Such methods include, but are not limited to, antigen-binding assays that are well known in the art, such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry (IHC). Advantageously in these embodiments anti-GPC4 sdAbs as herein disclosed are linked to a diagnostic compound, in particular a detectable label, as previously described.

The present disclosure further provides for the use of a GPC4 sdAb as herein disclosed in the manufacture of a reagent for use in a method of diagnosing a subject suffering from a GPC4-associated disease (e.g., a cancer as previously defined); the method comprising: determining the presence or expression level of GPC4 in a sample obtained from the subject by contacting the sample with an anti-GPC4 sdAb as herein disclosed and detecting the presence of the bound sdAb.

In another embodiment, the present disclosure provides an in vitro method for identifying a subject suffering from a GPC4-associated disease (e.g., a cancer), who is likely to respond to a treatment, such as an anti-GPC4 therapy, the method including: determining the presence or expression level of GPC4 in a (test) sample obtained from the subject by contacting the sample with an anti-GPC4 sd Ab as herein disclosed and detecting the presence of the bound sdAb, wherein the presence or expression level of GPC4 in said sample indicates that the subject is likely to respond to the treatment. Optionally, the expression level of GPC4 can be quantified and compared to a reference value as previously defined. The reference value is typically a threshold value, wherein a GPC4 expression level in the tested sample above the threshold value means that the subject is more likely to respond to treatment with the anti-cancer therapy. In one embodiment, the present disclosure also provides an in vitro method for predicting the responsiveness of an individual suffering from a cancer to a treatment with an anti-cancer therapy. Typically, the anti-cancer therapy is an anti-GPC4 therapy and comprises an anti-GPC4 antibody or a variant thereof, notably an anti-GPC4 sdAb as herein described (for example conjugated to a cytotoxic moiety), a multivalent binding compound or a chimeric GPC4 antigen receptor (CAR) as notably previously herein defined. The method comprises: determining the presence or expression level of GPC4 in a (test) sample obtained from the subject by contacting the sample with an anti-GPC4 sdAb, as herein disclosed and detecting the presence of the bound sdAb, wherein the presence or expression level of GPC4 in the tested sample indicates that the subject is more likely to respond to treatment with the anti-cancer therapy. Optionally, the expression level of GPC4 can be quantified and compared to a reference value as previously defined. The reference value is typically a threshold value, wherein a GPC4 expression level in the tested sample above the threshold value means that the subject is more likely to respond to treatment with the anti-cancer therapy.

The present disclosure also encompasses an in vitro method for monitoring a treatment efficacy in a patient receiving a treatment for a GPC4-associated disease, notably a cancer as previously defined. Typically, the treatment is a GPC4 antibody, notably an anti-GPC4 sdAb or a variant thereof as herein disclosed. Said method comprises determining in a biological sample of the patient at two or more time points the GPC4 protein level of expression (or GPC4 concentration). The determination of level of expression of the GPC4 protein in a biological sample of the subject typically comprises: contacting a biological sample obtained from the subject with an anti-GPC4 single domain antibody as herein disclosed; determining the level of expression of GPC4 in said sample by detecting the binding of said anti-GPC4 sdAb to GPC4 expressed by the sample; and comparing the level of expression of GPC4 in said sample with a reference value.

When the determination of level of expression of the GPC4 protein in a biological sample of the subject is repeated, the reference value is typically the level of GPC4 expression determined at the prior time point.

Measurement of a higher GPC4 protein level of expression (typically assessed by determining GPC4 concentration in said biological sample) in a biological sample of the patient at a later time point, compared to a value obtained in a biological sample of the patient at an earlier time point (thus typically used as a reference value), is indicative that the patient is non-responder to the treatment. Measurement of a lower GPC4 protein level expression (or GPC4 concentration) is indicative that the patient is responding to the treatment. Measurement of an equal GPC4 protein level (or GPC4 concentration) at the two or more times points indicates that the GPC4 associated disease, typically the cancer, does not progress (z.e., is stable) in the patient.

In one embodiment, the present disclosure provides a method for monitoring a treatment efficacy in a patient receiving a treatment for a GPC4 associated disease, which further comprise the steps of:

(a) quantifying the concentration of GPC4 in a first biological sample of a patient, typically using a GPC4 sdAb as herein disclosed, and optionally comparing the obtained value with a reference value and determining whether the obtained value is increased, decreased or is stable with regard to the reference control value;

(b) providing a treatment to the patient, typically when said obtained value is increased with regard to the reference value;

(c) quantifying again the concentration of GPC4 in a second biological sample from said patient (after having provided the treatment, notably after having initiated the treatment) and comparing the obtained concentration with the value prior to treatment obtained from the first biological sample (typically the value obtained at step a)) and optionally to another corresponding control value or to the reference value.

Steps b) and c) or the method can be repeated several times, notably 2 to 3 times, during the treatment and after treatment to monitor any disease recurrence,

Typically, the treatment is a GPC4 antibody, notably an antiGPC4 sdAb or a variant thereof as herein disclosed, such as an anti-GPC4 sdAb conjugate, a multivalent binding compound or a chimeric GPC4 antigen receptor as herein disclosed notably in the method of treatment section.

The present disclosure also provides a kit to perform any of the diagnosis or monitoring methods as above mentioned, and comprising one or more anti-GPC4 sdAbs as herein disclosed as well as suitable reagents.

In the following, the invention will be illustrated by means of the following examples and figures. Table 2: Sequences of the present disclosure

CDRs are numbered according to the IMGT nomenclature.

FIGURES LEGENDS Figure 1: Identification of hGPC4-specific single-domain antibodies by phage display. (A) ELISA of the sixteen potential GPC4 binders selected on the recombinant hGPC4-Fc protein. (B) Amino acid sequences of these sixteen selected Nbs The complementary determining regions (CDR1, CDR2 and CDR3 were assigned according to the AbM definitions (Kontermann, R. E., and Dubel, S. (2014) Antibody Engineering, Volume 2, Springer Berlin, Berlin) and are shown with a grey background. According to the amino acid sequences the Nbs belong to four different groups named as RBI, RB2, RB3, vRB3. Note that RB3, vRB3 differs for Y to N mutation in the first amino acid of the FR3 domain. (C) FACS analysis of binding properties of one representative Nb belonging to the RBI, RB2 and RB3 classes to HeLa cells transfected with hGPC4 expression vectors (black curve), and to HeLa cells transfected with a non-specific plasmid (grey curve). Shaded grey areas show the signal given by an irrelevant Nb signal on the GPC4 transfected HeLa cells. (D) Binding of RB3 to recombinant hGPC4-Fc protein (black curve) and to hlgGs (grey curve) examined by ELISA. RB3 was added at increasing concentrations. One representative curve is shown. Apparent KD, shown on the side of the graph, were calculated by using the GraphPad Prism version 8 software, and they are presented as mean+SEM of n=2 biological replicates. (E) Binding of RB I to HeLa cells expressing either hGPC4 (black curve) or an irrelevant protein (cDNA Ctrl; grey curve) measured by flow cytometry. Cells were incubated with increasing concentrations of RB 1. Data were normalized by the values obtained with a not relevant Nb. One representative curve is shown. As above, apparent KD (KD), shown on the side of the graph, were calculated by using the GraphPad Prism version 8 software, and they are presented as mean+SEM of n=2 biological replicates..

Figure 2: Binding properties of the bivalent RBl-Fc Nb. (A) Schematic representation of the bivalent RBl-FcNb generated by genetically cloning the VHH domain of RBI in frame with the Fc domain of a human IgGl heavy chain (Fc). (B) FACS analysis of cell binding on HeLa cells transfected with hGPC4 (black curve) or with an irrelevant cDNA (cDNActrl; grey curve). Cells were incubated with increasing concentrations of RB l-Fc. Data are presented as mean+SEM of n=3 biological replicates. Before pooling, data were normalized by the values of the negative control obtained with a not relevant Nb-Fc. Apparent KD (KD) were calculated by using the GraphPad Prism version 8 software and reported below the graph. (C) Graph reporting qRT-PCR analyses of GPC4 mRNA levels in HeLA, MKN, SNU-449 cancer cells and in hiPSCs and hiPSCs with reduced GPC4 levels. Note the different GPC4 levels in cell lines. Data are presented as mean+SEM n=2 biological replicates. One-way ANOVA: ***£><0.001. (D) Immunofluorescence analysis of GPC4 in different cell lines by using RB1- Fc. An irrNb-Fc was used as negative control. RB l-Fc detected hGPC4 in HeLa cells transfected with hGPC4 (HeLa + GPC4) as well as endogenous hGPC4 in cells such as MKN and hiPSCs producing relatively high GPC4. No staining was detected in HeLa cells or hiPSCs GPC4sh due to their lack or low GPC4 transcript levels, respectively (see panel (C)). A commercially available mGPC4Ab was used to detect GPC4 in HeLa cells transfected with hGPC4 (HeLa + GPC4) and not transfected (HeLa). (E) FACS analysis of RBl-Fc cell binding on HeLa, MKN, SNU-449 cancer cells, and on hiPSCs and hiPSCs with reduced GPC4 levels. HGPC4 expression levels in different cell lines were evaluated by comparing the fluorescence staining given by RBl-Fc with that of a not relevant Nb-Fc (irrNb-Fc). (F) FACS analysis of RBl-Fc binding to hiPSCs and hiPSCs with reduced GPC4 levels applied at different concentrations. Data are presented as mean+SEM of n=3 biological replicates. Before pooling, data were normalized by the values of the negative control obtained with a not relevant Nb-Fc.

Figure 3: RBl-Fc targets a conformational epitope of hGPC4. (A-B) Immunoprecipitation assay with cell extracts from HeLa cells not transfected or transfected with hGPC4-HA tag. Protein extracts were prepared by using native (A) or denaturing (B) buffers. Immunoprecipitations were done by using RB 1-FC, a not relevant Nb-Fc (irrNb-Fc), and a commercially available mAbGPC4. Immunoprecipitated proteins were detected by western blot by using Anti HA antibodies. Note that native hGPC4 was immunoprecipitated from native cell lysates incubated with RBl-Fc and not with the mAbGPC4 or the irrNb-Fc control (A). In contrast to the mAbGPC4, RB 1-Fc is unable to immunoprecipitate hGPC4 from a denaturated cell lysate hGPC4 (B).

Figure 4: RBI -Fc elicits hGPC4-blocking activity. (A) Graph reporting viability of hiPSCs in the presence of increasing concentration of RB 1-Fc and irrNb-Fc. Cell numbers were measured in a metabolic activity-based cell viability assay. Data are presented as mean+SEM of n=3 biological replicates. Note that not significant RB l-Fc toxic effects were observed at concentrations up to 500nM. (B) Schematic representation of the endoderm differentiation protocol of hiPSCs applied to test the RB l-Fc blocking activity on 11GPC4. hiPSCs are plated at dayO and exposed to ACTIVIN A (Act A) and ACTIVIN A + 0.2%FBS (ActA+FBS) at day! and day 2 of differentiation. Endoderm differentiation is analyzed at day 3 by following the number of SOX17 positive cells. (C) Representative images of SOX17 positive cells in hiPSCs and hiPSCs with reduced GPC4 levels (GPC4sh hiPSCs) after 3 days of differentiation showing the increased distribution of endodermal cells in the GPC4sh hiPSC line. (D) Representative images of SOX17 positive cells in hiPSCs exposed to an irrNb-Fc control and to RBl-Fc at 50 and 500nM. hiPSCs were treated with the Nbs either from dayl of differentiation or starting from dayO. Note the increased distribution of SOX17 positive cells in hiPSCs treated with RB 1- Fc comparable to that observed in the GPC4sh hiPSC line (C). (E) Quantitative analysis of SOX17 positive cells in hiPSCs treated with an irrNb-Fc control and with RB l-Fc at 500nM. Note a ~10 % increase of SOX17 positive cells in differentiating hiPSCs incubated with RBl- Fc in comparison to hiPSCs incubated with an irrelevant Nb. Also, hiPSCs exposed to RBl-Fc at dayO have an higher trend for generating SOX17 positive cells in comparison to cells treated with RB 1-Fc starting from dayl. Data are presented as mean+SEM of n=2 biological replicates. One-way ANOVA: **p<0.01.

EXAMPLES

Materials and Methods

Cell Culture and cell transfection hiPSCs (human induced pluripotent stem cells) (Kim J, Magli A, Chan SSK et al. Expansion and Purification Are Critical for the Therapeutic Application of Pluripotent Stem Cell-Derived Myogenic Progenitors. Stem Cell Reports 2017;9(l):12-22) were maintained as previously described. HEK 293 (ATCC), Hela (ATCC), Huh-7 (ATCC) and SNU-449 (ATCC) were grown as monolayers in Dulbecco’s modified Eagle’s medium (Gibco, ref 61965-026) supplemented with 10% fetal bovine serum (Sigma- Aldrich, ref F9665), 1% Penicillin- Streptomycin (Gibco, ref 15140122) and 1 mM Sodium Pyruvate (Gibco, ref 11360070) and MKN-45 (ATCC) in Roswell Park Memorial Institute- 1640 medium (Gibco, ref) supplemented with 10% foetal bovine serum, 1% Penicillin- Streptomycin and 1 mM Sodium Pyruvate, at 37°C with 5% CO2.

For transfection assay, cells were seeded the day before transfection and transfected at 70%confluency. Transfection of the plasmids encoding hGPC4 (Sino Biological, ref HG10090- UT), hGPC4 HA-tagged (Sino Biological, ref HG10090-CY) and mouse TIGIT Flag-tagged Flag (Sino Biological, ref MG50939-NF) was done with Lipofectamine3000 (Invitrogen, ref L300-015) according to manufacturer’s instruction. Cells were harvested fixed 48 hours after the transfection. Efficient expression of hGPC4 and hTIGIT were analysed by immunocytochemistry by using mouse anti-GPC4 (1:200, TableSl) and mouse anti-FLAG (1:1000, Table SI) antibodies. Immunization of llama with GPC4-enriched cell membranes and nanobody (Nb) library (i.e.: single domain antibody) construction

The human GPC4 antigen was prepared by transfecting HEK293T cells with an expression plasmid encoding for the human GPC4 fused to an HA-tag (Sino Biological, ref HG10090-CY) with lipofectamine 3000 following manufacturer’s instructions. Two days after transfection, cells were lysed with a native lysis buffer (lOmM Tris, ImM EDTA and 5mM MgC12 5mM) to preserve the conformation of the GPC4 membrane proteins. Total cell membranes were prepared by ultracentrifugation and resuspended in PBS. Llama immunizations were executed in strict accordance with good animal practices, following the EU animal welfare legislation law and were approved by local authorities (French Ministry of Higher Education for Research and Innovation). An alpaca (Ardeche Lamas) was injected subcutaneously four times with membranes prepared from 1.3 x 10⁸ HEK293T cells transfected with human GPC4 on days 0, 9, 18, 28. On day 28 (P4) and day 42 (P5) blood was collected for lymphocyte preparation.

Two Nb libraries were constructed as described in Behar et al. 2009 (Behar G., Chames P., Teulon I., Comilion A., Alshoukr F., Roquet F., et al. . (2009). Llama single-domain antibodies directed against nonconventional epitopes of tumor-associated carcinoembryonic antigen absent from nonspecific cross -reacting antigen. FEBS J. 276, 3881-3893). In brief, total RNA from peripheral blood lymphocytes were used as template for first-strand cDNA synthesis with the oligo . Using this cDNA, the Nb- encoding sequences were amplified by two successive PCR rounds, digested with Bgll and Notl, and cloned into the Sifl and Notl sites of the phagemid vector pHEN-phoA-8HisGS (plasmid pHEN-phoA-8hisGS derived from pHEN phoa6his: Behar et al. 2009; Library diversities were of 1,59.10⁹ transformants for P4 and 4,16.10⁸ transformants for P5.

Phage display and panning method

The two bacterial libraries, P4 and P5, were mixed and grown in 50mL of 2YT medium containing lOOpg/mL ampicillin at 37 °C with shaking at 230rpm. When bacteria reached ODeoo between 0.4-0.6, they were infected with the KM13 (production system) helper phage using a multiplicity of infection of 5xl0⁹ pfu/mL for 30min at 37 °C without shaking. The culture was centrifuged for 15min at 3000xg, and bacterial pellet was re-suspended in 250ml of 2YTA with kanamycine (lOOpg/mL) for an overnight phage-nanobodies production at 30 °C with shaking. After cells centrifugation, the supernatant was collected to precipitate the phages with 1/5 (vol/vol) cold solution of 20% PEG8000 and 2,5 mM NaCl 1 hour at 4°C. After incubation, phages were spun down and resuspend with ImL of DPBS IX. A second round of precipitation was performed for 30min at 4°C. The phage pellet was resuspended with DPBS IX and stored at -80 °C with 20 %glycerol.

To avoid non-specific selection four round of library panning were performed. The antigen used for panning was the purified recombinant hGPC4 fused to the Fc region of hlgGl (hGPC4-Fc; R&D Systems, ref 9195-GP-050). A 96-well ELISA plate (Maxisorb; Nunc/Thermo Fisher Scientific, ref 442404) was used to capture 4,4pg of hGPC4-Fc in DPBS IX (0,4pg per well) at 4°C overnight with shaking. After the coating, the plate was treated with blocking buffer [3%(wt/vol) milk in DPBS IX] at room temperature for Ihour with shaking. After two washes with DPBS IX containing 0.1%Tween-20 and two washes with DPBS IX, the Fc tag of the recombinant protein hGPC4-Fc was masked by 190pg of a solution of Fc tag-specific Nbs in blocking buffer ( 19pg per well) at room temperature for Ihour with shaking. The phage library, previously precipitated to remove glycerol, was resuspended in ImL blocking buffer and incubated for 90min at room temperature with the GPC4 antigen. After nine washes with DPBS IX containing 0.1%Tween-20 and three washes with DPBS IX, bound phages were eluted by treatment with 1 mL of Trypsin (Sigma- Aldrich, ref T1426) at 1 mg/mL in DPBS IX for 30min at room temperature. Phages were rescued and reamplified by infection of TGI (production system) and phage production as above, and used for the other rounds of panning. To maximize the selection of GPC4 binders a depletion step was added before the third and fourth panning on plates coated with hlgGl (l lpg; R&D Biosystem, ref 1-001-A) to deplete Fc binders. Finally, to obtain hGPC4 specific clones, 180 single bacteria colonies were picked and screened by ELISA for specific binding to the hGPC4-Fc protein. The VHH/VH genes of the selected Nbs were sequenced to characterize Nb diversity (GENEWIZ).

Expression and purification of recombinant Nbs

Following sequencing a representative Nb for each group underwent purification. The pHEN- phoA-8HisGS plasmid harboring Nb genes were transformed in BL21 E. coli strain (production system). The transformed bacteria were grown in 2YT medium and spread at low density on agar plates to isolated single clones. Single fresh clones were picked from agar plates, inoculated into 2YT medium supplemented with lOOpg/mL ampicillin and 2% glucose and grown at 37 °C overnight to generate large scale bacteria cultures. To ensure Nb monoclonality, four isolated clones were picked. Part of the culture was used for Nb sequence analysis. For the rest, when the cultures cells were reached ODeoo between 0.5-0.8, protein expression was induced with 100 p M isopropyl-b-D-thiogalactopyranoside overnight at 30 °C with shaking. Bacteria were then harvested and frozen at -20 °C. The expressed Nbs were extracted from the periplasm by osmotic shock with Bugbuster buffer (Novagen, ref 70584) supplemented with 20 pg/ml Lysozyme (Eurobio, ref GEXLYS00-6Z) and 25 U/pl Benzonase (Millipore, ref 70746) and purified by incubation with TALON Superflow (GE Healthcare, ref 28-9575-02) prior equilibration with DPBS IX. After incubation, the resin-Nbs complex was pelleted and resuspended with 5 volumes of buffer DPBS IX with 300mM NaCl. The solution was loaded on a column (Biorad, ref 731-1553) and the column was washed first with 5 column volumes of the same buffer and then with 10 column volumes of DPBS IX. The Nbs were eluted by fractions with DPBS IX with Imidazole 150mM (Sigma- Aldrich, ref 1202) and 300 mM NaCl. Nb enriched fractions were identified by using a Bradford protein assay (Bio-Rad). Finally, the Nbs were desalted to DPBS IX on PD-10 (GE Healthcare, ref 17085101) and concentrated by using Vivaspin (Sartorius, ref VS0611). Protein concentration was measured by Direct-Detect (Millipore). The integrity and purity of the Nbs were analyzed by protein gel and Western Blot.

Generation of the Nb-Fc construct, production and purification

To construct a Nb-Fc, the Nbs coding sequence were first amplified from the phagemid by PCR Phusion (Thermo Fisher Scientific, ref F-530L) with the oligos 5’HLSecVHH et 3’VHHEndH Then, the PCR products were cloned into the hlgGl Fc containing expression vector pHLSec- GiG4-Fc-6His (Aricescu et al. 2006; pre-digested with

Agcl/B stell by using the NEB HiFi kit according to the manufacturer’s instruction (New England Biolabs, ref E2621). Following plasmid transformation into DH5a E. coli bacteria (Invitrogen, ref 18263012), single colonies were grown in 2YT medium supplemented with 100 pg/mL ampicillin at 37 °C overnight. Four individual clones were picked to ensure monoclonality of the Nb-Fc following DNA sequence of the isolated plasmids. A large-scale culture was carried out to make a maxipreparation of Nb-Fc plasmid with EndoFree Plasmid Maxi kit according to the manufacturer’s protocol (Qiagen, ref 12362). The Nb-Fc plasmid was then transfected in Expi-293F cells (production system) (Gibco, ref A14635) following manufacturer’s protocol to produce the Nb-Fc proteins. Four days after transfection, the cell supernatant was collected, centrifuged and exposed to two rounds of dialysis (12-14 kDa cutoff, SpectrumLabs, ref 132678). Then, the Nb-Fc proteins were purified by taking advantage of their 6His tag and by using the TALON Superflow, as above. Purified proteins were snap- frozen and stored at -80°C.

ELISA

Maxisorb 96-well plates were coated either with hGPC4-Fc or with hErbB2-Fc (Sino Biological, ref 10004-H02H) or with hlgG were adsorbed at 4°C overnight onto 96-well immunoplates (Maxisorb;Nunc) at 0,4pg per well as above. All ELISA steps were subsequently done at room temperature. The plate was treated with blocking buffer [3% (wt/vol) milk in DPBS IX] for Ih with shaking and washed three times with DPBS IX containing 0.1%Tween- 20. For screening, 70pL of supernatant from TGI culture was mixed to 3 Opl of blocking buffer and added to the plate for 1 hour. After incubation, the wells were washed 3 times with DPBS IX containing 0.1%Tween-20 and the bound Nbs were detected by addition of anti-6His peroxydase conjugate antibodies (1:5000; Table SI).

To measure the Nbs or Nbs-Fc affinities, the purified Nbs or Nbs-Fc were diluted in 3%(w/v) milk-DPBS IX and added at the various concentrations to the protein-coated wells for 1 hour, either in triplicates or duplicates and incubated for 1 hour. After incubation, the wells were washed 3 times with 0.1%Tween-20 in DPBS IX. The bound Nbs and Nbs-Fc were detected by the addition of either anti-6His peroxydase conjugate antibodies or goat anti-human peroxydase conjugate antibodies, respectively (1:5000; Table SI). Wells were then washed 3 times with 0.1% Tween-20 in DPBS IX and binding was revealed with TMB (Thermo Scientific, ref 34029). The reaction was stopped by addition of IM HC1 and the absorbance was read at 450nm using the ELISA plate reader TECAN. The coating of hGPC4-Fc protein was controlled by incubation with mouse anti-GPC4 antibodies (1:200, Table SI) and revealed with goat anti-Mouse peroxydase conjugate antibodies (1:5000; Table SI). The coating of human erbB2-Fc or hlgG proteins were revealed with goat anti-Human peroxydase conjugate antibodies.

Flow cytometry

For flow cytometry, growing cells were washed and harvested with Accumax. Following centrifugation to remove Accumax, cells were resuspended in ice-cold DPBS IX containing 5%(wt/vol) BSA (Euromedex, ref 04-100-812) and plated into 96well-plate at 1,5.10⁵ - 3.10⁵ cells per well (Nunc/Thermo Fisher Scientific, ref 24950). Cells were incubated with various amounts of Nbs or Nbs-Fc for 1 hour at 4°C and then washed twice with DPBS IX with 5% (wt/vol) BSA to remove not specific binding. When testing affinity of the unmodified Nbs, cells were incubated first with anti-His primary antibodies (1 : 1000, TableS 1) for 45min with shaking and then, following two washes with DPBS1X with 5%(wt/vol) BSA, with Alexa647conjugated goat anti-mouse secondary antibody (1:500 TableSl) for 30min. When testing affinity of Nbs-Fc, cells were incubated with Phycoerythrine conjugated goat antihuman secondary antibodies (1:250, Table SI) for 30min. Finally, cells were washed three times with DPBS1X and resuspended in a final volume of 100 pl DPBS1X. For each experiment, at least 10,000 cells were analyzed in a flow cytometer. The fluorescence associated with the live singlet cells was measured using a MACS Quant (Myltenyi).

Immunoprecipitation assays

Cells were lysed either with non-denaturing buffer following Abeam recommendations (20 mM of Tris-HCl, 137 mM of NaCl, 2 mM of EDTA and 1% of NP-40 in deionized water) or with the denaturing RIPA buffer (150mM sodium chloride, 1.0% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0 in deionized water) and the protein concentration was measured by Coomassie blue assay (Biorad, ref 5000006). 1,5 mg of cell lysate was incubated with 10 pg of indicated antibodies in 500pl of nondenaturing buffer and rotated overnight at 4°C. 30pl of packed protein A-Agarose beads (Amersham Pharmacia Biotech, ref 17096303) were equilibrated with non-denaturing buffer and added to sample. Following 2hours rotation at 4°C, beads were spun down and washed with non-denaturing buffer five times. The immune complexes were released from beads after 5min boiling in lOOpl of 6X loading buffer (Thermo Fisher Scientific, ref 26620). Western blot analysis was performed on 40 pl of immune complexes and hGPC4 was detected by using rat anti-HA antibody (1:1000, TableSl) according to standard procedure (Fico A, de Chevigny A, Egea J, Bosl MR, Cremer H, Maina F, Dono R. Modulating Glypican4 Suppresses Tumorigenicity of Embryonic Stem Cells while Preserving Self-renewal and Pluripotency. Stem Cells. 2012;30:1863-1874). 60 pg of the cell lysate input were included as control.

Immunocytochemical analyses on cultured cells

Cells grown on glass coverslips or labtek (Nunc/Thermo Fisher Scientific, ref 178599) were fixed for lOmin with 4% PFA in DPBS1X. All steps were done at room temperature unless otherwise specified. After three washes with PBS, fixed cells were permeabilized by a 20 minutes treatment with 0,1% TritonX-100 in DPBS1X for extra-cellular staining and 0,3% TritonX-100 in DPBS1X for intra-cellular staining. Following Ihour blocking in 3% BSA (Sigma-Aldrich, A9418), 2% Donkey serum (Millipore, ref S30), 0.3% or 0,1% TritonX-100, in DPBS1X, cells were incubated with primary antibody overnight at 4 °C in the same blocking solution. After three washes with 0.3% or 0,1% TritonX-100 in DPBS IX, cells were incubated with secondary antibodies (1:500 + DAPI 5 pg/ml) for 1 hour protected from light. Finally, after three washes with 0.3% or 0,1% TritonX-100 in DPBS1X and two rinses with DPBS1X, coverslips or Labtek were mounted by using ProLong™ Gold Antifade Mountant. The images were captured on a Zeiss Axiolmager APO Z1 microscope.

GPC4 quantitative PCR analysis

For GPC4 quantitative PCR, 2,7ng of cDNA was amplified by using SYBR Green qPCR SuperMix (Invitrogen, ref 11761) and O.lpM of forward and reverse primers. Levels of all transcripts (Ct) were normalized to those of the housekeeping gene GAPDH (ACt) and subsequently to the ACt of the A549 cell lines that do not express GPC4 (AACt). Results were reported as relative quantities (RQ = 2^A-AACt).

Cell viability assays

For survival assays, hiPSCs (human induced pluripotent stem cells) were seeded in 96 wellplates at 1.10⁴ cells per well. Two days after, the medium was changed and replaced with fresh medium containing increasing amount of RB 1-Fc. At the day three, cell viability was revealed by addition of CellTiter-Glo reagent (Promega, ref G7570) in cell supernatant. After 20 minutes of incubation at room temperature (light protected), the luminescence activity was analyzed with a luminometer microplate reader (Berthold). Cell survival was normalized to cells treated with vehicle (PBS).

Analysis of Nbs-Fc blocking properties: endoderm differentiation in hiPSCs

In vitro differentiation of hiPSCs into definitive endoderm progenitors was performed according to D’Amour et al 2005 (D’ Amour et al 2005. Nat. Biotechnol. 23, 1534-1541 2005). hiPSCs were exposed to increasing concentrations of the RBl-Fc Nb added in to culture medium. Two treatments were realized on hiPSC. For the first one, the RBl-Fc treatment was initiated the day of seeding (day 0). For the second one, the RB 1-Fc treatment was initiated the first day of differentiation (day 1). In both cases, differentiating cells were exposed to RBl-Fc through all differentiation procedure (3 days). The medium was changed daily to keep a constant level of RB 1-Fc. Then, cells were fixed and analyzed by immunocytochemistry for the presence of the SOX17 protein. The endoderm differentiation efficiency was determined by analyzing the percentage SOX17 positive cells over the DAPI positive nuclei.

Western Blot To determine the quality of Nbs and Nbs-Fc production, at least 1 pg of proteins were resuspended in 4X Laemli loading buffer (Bio-Rad, ref 161-0747) and loaded on a 4-15% mini- PROTEAN® TGX™ precast protein gels (Bio-Rad, ref 4568084). The gel was revealed with Image Analyzer (Gel doc EZ, Bio-Rad) and transferred to Nitrocellulose membrane (Bio-Rad, ref 1704270). Following blocking with 5%(wt/vol) BSA in DPBS1X for Ih at room temperature, membranes were incubated with anti-6His peroxidase conjugated antibodies (1:5000, Table SI) at room temperature for 30 minutes. A solution of DAB (Sigma- Aldrich, ref D5905) and H2O2 (Sigma- Aldrich, ref 95321) in DPBS1X was used to reveal peroxidase activity.

Statistical Analysis

Quantitative analyses of endoderm differentiation efficiency were performed manually with the FIJI image processing software (Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. 2012. Fiji: An open-source platform for biological- image analysis. Nat Methods 9: 676- 682.). Statistical analyses were performed using the most adapted test for each study (e.g. One way ANOVA) using the GraphPad Prism version 8 software. Nb-binding curves were plotted using nonlinear least square fit. Kd values were calculated by using the one binding site method reported in the Prism version 8 software. Statistic values were reported as: ns=not significant, * = p value < 0,05, ** = pvalue < 0,01, *** = pvalue < 0,001.

Table SI. List of all primary antibodies used.

Results

Generation of hGPC4-targeting Nbs

To identify antibodies specific for GPC4 in its native conformation, one llama (Llama glama) was immunized with membrane protein extracts of HEK cells overexpressing the hGPC4 protein fused to a carboxyterminal HA-tag. Two Nb phage display libraries were constructed by using pools of VHH/VH gene fragments amplified from RNA of blood lymphocyte taken at weeks 4 and 6 following immunization. The 2 libraries consisted of -9,93.10⁶ transformants each and -99% of transformants in each library harboured a vector with the right insert size. Phage particles were generated and subjected to panning on purified recombinant hGPC4-Fc, a fusion of the entire hGPC4 protein to the Fc region of human IgGl. After four consecutive rounds of panning and two rounds of depletion with human IgGl, 360 individual phage clones were isolated and screened by ELISA using recombinant hGPC4-Fc and colony supernatant. Sixteen potential hGPC4 binders were selected based on hGPC4-Fc OD values of at least twofold higher than that of hlgGs. (Figure 1A).

Gene sequencing of the selected Nbs revealed that all of them contained the three representative complementary-determining (CDR) regions, which are known to contribute to antigen-binding specificity. Moreover, based on sequence homology, they were classified in four groups named as RBI, RB2, RB3 and vRB3. Nbs belonging to RB3 and vRB3 most likely belonged to the same related B-cell clones as their amino acid sequences differ for a Y58 to N58 mutation in the first amino acid of the FR3 domain (Figure IB). One clone for each group was selected and transformed into BL21 E.coli cells to allow large-scale expression of the Nbs without the pill fusion (21). The expressed Nbs were recovered from the periplasmic extracts and purified.

Cell binding is one of the most important criteria for therapeutic antibodies targeting cellsurface proteins; therefore, the selected Nbs were subjected to cell-based screening assays in which their binding properties to cells expressing hGPC4 were assessed. As HeLa cells do not express detectable levels of GPC4, HeLa transfected with an hGPC4 expression vectors were used for Nb positive selection, whereas non-transfected and HeLa cells transfected with a nonspecific plasmid were used for negative selection. Nb binding specificity was established by comparing cell-associated fluorescence among different samples by flow cytometry. Results of this analysis revealed that the identified Nbs belong to three different groups. The Nbs of the RB 1 group bound only to HeLa cells expressing hGPC4 and not to negative controls (Figure 1C), and were classified as "specific binders of GPC4 recombinant protein and of cell expressed GPC4”. The Nbs of the RB2 group bound both to GPC4-expressing HeLa cells and to negative controls. They were classified as “non-specific binders", and excluded from further analysis (Figure 1C). By contrast, the Nbs of the RB3 and vRB3 group did not bind to any cell lines despite the fact they were selected for interaction with the recombinant hGPC4 protein. Therefore, they were classified as “specific binders of GPC4 recombinant protein and nonbinders of cell expressed GPC4” (Figure 1C).

Analysis of hGPC4 Nb-binders

To further characterize the binding properties of RB 1 and RB3, their apparent binding affinity for hGPC4 was determined. Affinity measurements were performed by using serial Nb dilutions of RB3 and RBI either by ELISA with purified recombinant hGPC4-Fc or by flow cytometry with HeLa cells expressing hGPC4, respectively. As controls for not specific binding ELISA on hlgGs and irrelevant Fc-fused recombinant protein were performed, as well as flow cytometry on HeLa cells and HeLa cells expressing an irrelevant protein. Quantification analysis from independent experiments revealed that RB3 binds purified hGPC4 with an affinity constant of 9, 1+1, 6 nM and RB 1 binds to cell-expressed GPC4 with an affinity constant of 83,5+42 nM (Figure IB and 1C) .. Taken together these results show that the inventors have successfully identified the first two anti-hGPC4 single-domain antibodies by phage display. GPC4-targeting RB I and RB3 correspond to the first GPC4-targeting Nbs (i.e., sdAb). Moreover, given the different binding capabilities of RB I and RB3, these Nbs might complement each other by enabling efficient hGPC4-targeting both in vitro and in human cells respectively. Finally, the interaction of RB I with native hGPC4 expressed by cells suggests potential blocking properties. Given this possibility, RBI was selected for further analysis.

Dimerization of the RBI Nbs enhances its hGPC4-binding capability

It has been shown that the genetic fusion of monovalent Nbs to the Fragment Crystallisable Region (Fc-fragment) of conventional antibodies can enhance Nb potencies and stability in biological fluids as well as endow them with properties such as translocation across the bloodbrain barrier and low cytotoxicity. To take advantage of these findings, the inventors genetically engineered RB I to generate a RBl-Fc fusion format. This was done by cloning the VHH domain of RB 1 in frame with the Fc domain of a human IgGl heavy chain. This resulted in a bivalent RB l-Fc dimer consisting of two 40kDa subunits, as compared to the 15 kDa size of the monovalent RBI Nb (Figure 2A).

The GPC4-binding properties characterising the bivalent RB l-Fc Nb was then tested by using different approaches. First, the inventors determined the apparent affinity constant of RB l-Fc by using as above HeLa cells expressing hGPC4 protein by flow cytometry in order to compare values with that of RB I. Quantitative analysis from independent experiments established that RBl-Fc Nb acquires ~15 fold increased in binding capability when compared to RBI, as shown by a shift of the apparent affinity constant from 83,5+42 nM for RBI to 5, 8+1, 9 nM for RB l- Fc.

Next, the inventors studied the ability of the generated RBl-Fc construct to bind and detect various levels of endogenously expressed hGPC4. By employing qRT-PCR analysis, they selected a panel of cell lines representing those with high hGPC4 protein levels such as the cancer cell line MKN and human induced pluripotent stem cells (hiPSCs), and those with low hGPC4 levels such as hiPSCs with down regulated GPC4 (obtained by using shRNA targeting approach) and the SNU-449 cancer line, respectively (Figure 2C). HeLa cells were used as negative control line (Figure 2C).

The capability of RB 1-Fc to bind hGPC4 exogenously and endogenously produced by cells was first tested by immunocytochemistry using different Nb concentrations ranging from 8 to 25nM. RBl-Fc detected the exogenously and endogenously produced hGPC4 in accordance with changes in protein levels, respectively (Figure 2D). In particular, RBl-Fc detected hGPC4 in HeLa cells transfected with a GPC4-Fc cDNA but not in un-transfected cells. Moreover, RB 1- Fc detected hGPC4 in cells with high transcript levels such as hiPSCs and MKN and not in cells with low or none GPC4 expression such as hiPSCs with down-regulated GPC4 and HeLa cells (Figure 2D). The ability of RBl-Fc to bind hGPC4 exogenously and endogenously produced by cells was then tested by flow cytometry to also have a quantitative readout. RB 1- Fc detected hGPC4 in cells in accordance with hGPC4 expression levels (Figure 2E). For example, the levels of hGPC4 protein detected in hiPSCs was at least 6-fold higher than those detected in hiPSCs with down-regulated GPC4 (hiPSCs: mean fluorescence at 133nM RBl-Fc = 39.5; hiPSC GPC4sh: mean fluorescence at 133nM RB l-Fc = 6.3; Figure 2F), which show a ~7-fold reduced hGPC4 transcript levels (hiPSCs: mean RQ 16108+1290,3; hiPSC GPC4sh: mean RQ 1970+110,1; Figure 2 C).

RBl-Fc recognize a conformational epitope of the native hGPC4

The above described results demonstrate that RB l-Fc can interact with the cell-expressed hGPC4 protein. To go further in the molecular interactions between RB l-Fc and hGPC4, the inventors tested RBl-Fc binding to native and unfolded hGPC4. This was first assessed by performing western blot analysis of cell extracts from HeLa cells transfected with hGPC4. In contrast to the commercially available anti-GPC4 monoclonal antibody used as control, neither RBI nor RB l-Fc recognized hGPC4 under reducing, none-native, conditions, thus indicating that RBl-Fc does not target a denatured hGPC4 protein. To explore this issue further, they performed immunoprecipitation experiments by using different cell lysis buffers. Results from these studies established that RBl-Fc interacts with hGPC4 only when hGPC4 is in its native conformation. Indeed, the use of lysis buffers containing ionic detergents, which can alter protein's charge and structure, drastically impairs the ability of RBl-Fc to immunoprecipitate cell-expressed hGPC4 (Figure 3B). In contrast, RB l-Fc immunoprecipitates cell-expressed hGPC4 only when the cells are lysed by using non-denaturing buffers (Figure 3A) These immunoprecipitation results also revealed that RB I -FC binds the hGPC4 core protein and not the heparan sulphate side chains present in hGPC4 and other HSPG proteins (Figure 3 A), which highlights the specificity of RB 1-FC for hGPC4 targeting.

RBl-Fc blocks hGPC4 biological functions As above discussed, impairing hGPC4 functions in hiPSCs and in cancer cell types such as pancreatic cancer stem cells can potentially provide a clinically relevant approach for improving hiPSC therapeutic applications as well as for the treatment of distinct cancer types. In this context, the availability of 11GPC4 blocking antibodies can be of great importance. The inventors therefore tested whether RBl-Fc inhibits hGPC4 functions.

As a first approach, they established whether RB 1-Fc elicits non-specific toxic effects on cells. Their loss-of-function studies in hiPSCs have shown that down regulation of hGPC4 in such cells types does not affect their survival. Therefore, RB l-Fc toxic effects were assessed by exposing hiPSCs to increasing concentrations of RBl-Fc and to an irrelevant Nb as negative control. Quantification analysis of surviving cells, done by using a metabolic activity-based cell viability assay, revealed that RB l-Fc does not elicit significant non-specific cytotoxic effects when applied to cells at concentrations below 500 nanomolar (Figure 4A).

Next, the inventors tested putative RB l-Fc neutralizing capacity. They have previously established that hiPSCs, in which hGPC4 proteins levels are reduced by means of shRNA targeting (GPC4sh), undergo a more efficient endoderm lineage entry than controls in response to endoderm triggering factors (unpublished results). This phenotype is apparent upon three days treatment of hiPSCs with ACTIVIN-A as evidenced by a ~ 10 to 15 fold increase in the numbers of SOX- 17 positive cells detected by immunocytochemistry (Figure 4B and 4C). They, therefore tested whether treatment of hiPSCs with the RB 1-Fc Nb is able to block hGPC4 activity in these cell types and induce differentiation properties, as assessed through a more efficient endoderm lineage entry. The ability of RBl-Fc to influence the endoderm differentiation of hiPSCs was tested by performing differentiation experiments in the presence of various concentrations of RB l-Fc or of an irrelevant Nb, as negative control. Nbs were applied at the concentrations of 50, 250 and 500nM, as they do not induce toxic effects on cells (Figure 4A). Moreover, the inventors performed two different treatments. In the first, RBl-Fc was applied at the onset of endoderm differentiation (dO; Figure 4B) and maintained throughout all differentiation procedure (Figure 4B). In the second treatment, hiPSCs were exposed to RB1-FC for 24 hours before the start of differentiation and then treated RBl-Fc during the overall differentiation experiment (Figure 4B). The rationale behind this latter approach was that down-regulation of hGPC4 in undifferentiated hiPSCs could induce new biological properties supporting a more efficient response to differentiation signals. Therefore, treatment of hiPSCs with RB l-Fc at undifferentiated stages should result in an hiPSC type wherein functional features are similar to hiPSCs wherein GPC4 is down-regulated. Results from these studies revealed that treatment of hiPSC with RBI -FC drastically increases their capability to enter the endodermal lineage as shown by the abundance of SOX17 positive cells (Figure 4D). Quantitative analysis showed that in a concentration ranging from 50 nM to 500nM RB 1-Fc is sufficient to promote a ~10 % increase of SOX17 positive cells in differentiating hiPSCs with respect to hiPSCs treated with the same concentration of an irrelevant Nb (Figure 4E). Moreover, this increased in SOX17 positive cells was comparable to that observed in hiPSCs with reduced hGPC4 protein levels (Figure 4C), thus highlighting that RB l-Fc is a hGPC4- antagonizing Nb. Interestingly, hiPSCs exposed to RBl-Fc for 24 hours before the start of differentiation displayed an higher trend for generating SOX17 positive cells in comparison to cells not treated at the undifferentiated stage (Figure 4D and 4E). These results suggest a treatment at undifferentiated stage further increases the potency of RB 1-Fc as an antagonist of hGPC4-biological functions. Altogether, these results define the Nb RB 1-Fc as the first hGPC4- blocking antibody. The use of RBl-Fc allows to block hGPC4 activity without the use of genetic manipulations. The above results also suggest that RB 1-Fc can be used to study hGPC4 functions in a temporal and spatial regulated manner.

References

Fico A, Maina F, Dono R. Fine-tuning of cell signaling by glypicans [in English]. Cellular and Molecular Life Sciences 2011;68(6):923-929.

2 Fico A, Dono R. Signalling mechanisms underlying congenital malformation: the gatekeepers, glypicans. In: Congenital Malformations.: Alastair Sutcliffe, University College London, University of London, United Kingdom, 2011.

3 Poulain FE, Yost HJ. Heparan sulfate proteoglycans: a sugar code for vertebrate development? Development 2015;142(20):3456-3467.

4 Matsuo I, Kimura- Yoshida C. Extracellular distribution of diffusible growth factors controlled by heparan sulfate proteoglycans during mammalian embryogenesis. Philos Trans R Soc Lond B Biol Sci 2014;369(1657).

5 Yan D, Lin X. Shaping morphogen gradients by proteoglycans. Cold Spring Harb Perspect Biol 2009;l(3):a002493.

6 Kakugawa S, Langton PF, Zebisch M et al. Notum deacylates Wnt proteins to suppress signalling activity. Nature 2015;519(7542):187-192. Topczewski J, Sepich DS, Myers DC et al. The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension. Dev Cell 2001 ;1(2):251-264.

8 Galli A, Roure A, Zeller R et al. Glypican 4 modulates FGF signalling and regulates dorsoventral forebrain patterning in Xenopus embryos. Development 2003;130(20):4919-4929.

9 Jakubovic BD, Jothy S. Glypican-3: from the mutations of Simp son- Golabi-Behmel genetic syndrome to a tumor marker for hepatocellular carcinoma. Exp Mol Pathol 2007;82(2):184-189.

10 Li N, Gao W, Zhang YF et al. Glypicans as Cancer Therapeutic Targets. Trends Cancer 2018;4(ll):741-754.

11 Nishida T, Kataoka H. Glypican 3-Targeted Therapy in Hepatocellular Carcinoma. Cancers (Basel) 2019; 11(9).

12 Li N, Fu H, Hewitt SM et al. Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma. Proc Natl Acad Sci U S A 2017;114(32):E6623-E6631.

13 Dono R. Glypican 4 down-regulation in pluripotent stem cells as a potential strategy to improve differentiation and to impair tumorigenicity of cell transplants. Neural Regen Res 2015;10(10):1576-1577.

14 Fico A, De Chevigny A, Egea J et al. Modulating Glypican4 suppresses tumorigenicity of embryonic stem cells while preserving self-renewal and pluripotency [Research Support, Non-U. S. Gov't] [in eng]. Stem Cells 2012;30(9):1863-1874.

15 Fico A, de Chevigny A, Melon C et al. Reducing Glypican-4 in ES Cells Improves Recovery in a Rat Model of Parkinson's Disease by Increasing the Production of Dopaminergic Neurons and Decreasing Teratoma Formation [in English]. Journal of Neuroscience 2014;34(24):8318-8323.

16 Fang Y, Shen ZY, Zhan YZ et al. CD36 inhibits beta-catenin/c-myc-mediated glycolysis through ubiquitination of GPC4 to repress colorectal tumorigenesis. Nat Commun 2019;10(l):3981. 17 Cao J, Ma J, Sun L et al. Targeting glypican-4 overcomes 5-FU resistance and attenuates stem cell-like properties via suppression of Wnt/beta-catenin pathway in pancreatic cancer cells. J Cell Biochem 2018; 119(11):9498-9512.

18 Tamori Y, Kasuga M. Glypican-4 is a new comer of adipokines working as insulin sensitizer. J Diabetes Investig 2013;4(3):250-251.

19 Yoo HJ, Hwang SY, Cho GJ et al. Association of glypican-4 with body fat distribution, insulin resistance, and nonalcoholic fatty liver disease. J Clin Endocrinol Metab 2013;98(7):2897-2901.

20 Kaplon H, Reichert JM. Antibodies to watch in 2019. MAbs 2019;l l(2):219-238.

21 Hamers-Casterman C, Atarhouch T, Muyldermans S et al. Naturally occurring antibodies devoid of light chains. Nature 1993;363(6428):446-448.

22 Muyldermans S. Nanobodies: natural single-domain antibodies. Annu Rev Biochem 2013;82:775-797.

23 Khodabakhsh F, Behdani M, Rami A et al. Single-Domain Antibodies or Nanobodies: A Class of Next- Generation Antibodies. Int Rev Immunol 2018;37(6):316-322.

24 De Genst E, Silence K, Decanniere K et al. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci U S A 2006;103(12):4586-4591.

25 Feng M, Gao W, Wang R et al. Therapeutically targeting glypican-3 via a conformationspecific single-domain antibody in hepatocellular carcinoma. Proc Natl Acad Sci U S A 2013;110(12):E1083-1091.

Claims

1. An anti-GPC4 single domain antibody (sdAb), wherein said anti-GPC4 sdAb has the following formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and wherein the CDRs are selected from:

- a CDR1 of SEQ ID NO: 1; a CDR2 of SEQ ID NO:2 and a CDR3 of SEQ ID NOG, or

- a CDR1 of SEQ ID NO:4; a CDR2 of SEQ ID NOG and a CDR3 of SEQ ID NOG.

2. The anti-GPC4 sdAb according to claim 1 having a sequence set forth in SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 19.

3. The anti-GPC4 sdAb according to claim 1 or 2, which is linked directly or indirectly, covalently or non-covalently to a compound of interest selected from a nucleic acid, a polypeptide or a protein, a virus, a toxin and a chemical entity optionally wherein said sdAb is linked directly or indirectly, covalently or non-covalently to a diagnostic compound selected from an enzyme, a fluorophore, an NMR or MRI contrast agent, a radioisotope and a nanoparticle; optionally wherein said sdAb is linked directly or indirectly, covalently or non-covalently to a therapeutic compound selected from cytotoxic agents, chemotherapeutic agents, radioisotopes, targeted anti-cancer agents, immunotherapeutic agents (such as immunosuppressants or immune stimulators), and lytic peptides..

4. A humanized anti-GPC4 sdAb having:

- a CDR1 of SEQ ID NO:1; a CDR2 of SEQ ID NOG and a CDR3 of SEQ ID NOG;

- a CDR1 of SEQ ID NOG; a CDR2 of SEQ ID NOG and a CDR3 of SEQ ID NOG; a sequence of SEQ ID NO: 15; a sequence of SEQ ID NO: 16; a sequence of SEQ ID NO: 19; a sequence having at least 90 % identity with SEQ ID NO: 15 a sequence having at least 90 % identity with SEQ ID NO: 16; a sequence having at least 90 % identity with SEQ ID NO: 19;

- a CDR1 of SEQ ID NO: 1 ; a CDR2 of SEQ ID NOG and a CDR3 of SEQ ID NOG and further having one or more conservative amino acid modifications in one or more of these CDRs; or and further having one or more conservative amino acid modifications in one or more of these CDRs.

5. A multivalent binding compound comprising at least a first sdAb consisting in an anti-GPC4 sdAb as defined any one of claims 1 to 4, and further comprising at least a second sdAb, wherein said second sdAb binds to the same or to a second antigen; optionally wherein, the second sdAb is an anti-GPC4 sdAb as defined in any one of claims 1 to 4; optionally wherein, the first and at least second sdAbs are in a tandem format; notably wherein the first and second sdAbs are constructed in a head to tail tandem format

6. A multivalent binding compound according to claim 4, which is in a sdAb-Fc format; optionally wherein the multivalent binding compound is in a bivalent sdAb-Fc format; optionally wherein, the bivalent sdAb-Fc compound comprises a first and a second sdAb as defined in any one of claims 1 to 6 and which are identical.

7. A chimeric antigen receptor (CAR) comprising (a) an antigen binding domain comprising a sdAb consisting in an anti-GPC4 sdAb as defined in any one of claims 1-4, or a multivalent binding compound as defined in any one of claim 5 or 6, (b) a transmembrane domain; and (c) an intracellular domain.

8. An isolated nucleic acid comprising a nucleic acid sequence encoding a humanized sdAb directed against GPC4 as defined in any one of claims 1-4, a multivalent binding compound as defined in any one of claim 5 or 6 or a CAR as defined in claim 7.

9. A vector comprising a nucleic acid according to claim 8.

10. A host cell comprising a nucleic acid according to claim 8, or a vector according to claim 9.

11. An isolated cell or population of cells expressing an anti-GPC4 SdAb as defined in any one of claims 1-4, a multivalent binding compound as defined in any one of claim 5 or 6, or a

CAR as defined in claim 7 ; optionally wherein the cell is an immune cell; optionally wherein the immune cell is selected from macrophages, NK cells, CD4+/CD8+, TILs/tumor derived CD8 T cells, central memory CD8+ T cells, Treg, MAIT, and Yδ T cells.

12. The anti-GPC4 SdAb of claims 1-4, the multivalent binding compound of claims 5-6, the CAR of claim 7, the nucleic acid of claim 8, the vector of claim 9, the host cell of claim 10, or the isolated cell or cell population of claim 11, for use in therapy, optionally for use in the treatment of a disease selected from a proliferative disease, or a metabolic disorder or for cellbased replacement therapy in a subject in need thereof, optionally wherein the disease is a GPC4 associated disease, notably a GPC4 associated cancer.

13. Use of an anti-GPC4 SdAb as defined in any one of claims 1-4 for the detection of GPC4 in a biological sample.

14. Use of an anti-GPC4 sdAb as defined in any one of claims 1-4 or of a multivalent binding compound as defined in any one of claims 5-6, in vitro or ex vivo, to promote selfrenewal and differentiation of stem cells (SCs); optionally wherein stem cells (SCs) are embryonic stem cells or pluripotent stem cells, such as induced PSCs and notably human iPSCs; optionally wherein said sdAb or multivalent binding compound promotes efficient lineage entry, notably in mesoderm, ectoderm, or endoderm lineage.

15. A method of producing differentiated stem cells (SCs), notably midbrain dopaminergic neurons (mDA neurons), comprising culturing said SCs in the presence of an anti-GPC4 sdAb as defined in any one of claims 1-4 or of a multivalent binding compound as defined in any one of claims 5-6;

Optionally wherein said SCs are pluripotent stem cells (PSCs), notably human induced PSCs (hiPSCs)

16. The anti-GPC4 SdAb of claims 1-4, the multivalent binding compound of claims 5-6, the CAR of claim 7, the nucleic acid of claim 8, the vector of claim 9, the host cell of claim 10, or the isolated cell or cell population of claim 11 for use as a medicament.

17. An in vitro method for diagnosing a GPC4-associated disease in a subject, wherein the method comprises: contacting a biological sample obtained from the subject with an anti-GPC4 single domain antibody as defined in to any one of claims 1-4; determining the level of expression of GPC4 in said sample by detecting the binding of said anti-GPC4 sdAb to GPC4 expressed by the sample; and comparing the level of expression of GPC4 in said sample with a reference value.

18. An in vitro method for determining the eligibility of a subject to a treatment with an anti-GPC4 therapy, wherein the method comprising: determining the presence or expression level of GPC4 in a sample obtained from the subject by contacting the sample with an anti- GCP4 sdAb as defined in any one of claims 1-4 and detecting the presence of the bound sdAb; optionally wherein the anti-GPC4 therapy is an anti-GPC4 sdAb as defined in any one of claims 1-4, a multivalent binding compound as defined in claims 5-6, or a chimeric antigen receptor as defined in claim 7.

19. An in vitro method for monitoring a treatment efficacy in a subject receiving a treatment for a GPC4-associated disease, wherein the method comprises determining in a biological sample of said subject, at two or more time points, the level of expression of the GPC4 protein, and wherein the determination of level of expression of the GPC4 protein in a biological sample of the subject comprises: contacting a biological sample obtained from the subject with an anti-GPC4 single domain antibody as defined in any one of claims 1-4; determining the level of expression of GPC4 in said sample by detecting the binding of said anti-GPC4 sdAb to GPC4 expressed by the sample; and comparing the level of expression of GPC4 in said sample with a reference value.

20. A method for identifying a subject suffering from a GPC4-associated disease, who is likely to respond to a treatment, the method including: determining the presence or expression level of GPC4 in a sample obtained from said subject by contacting the sample with an anti- GPC4 sdAb as defined in any one of claims 1-4, and detecting the presence of the bound sdAb, wherein the presence or expression level of GPC4 in said sample indicates that the subject is likely to respond to the treatment.

21. A method for predicting the responsiveness of an individual suffering from a cancer to a treatment with an anti-cancer therapy comprising: determining the presence or expression level of GPC4 in a sample obtained from said subject by contacting the sample with an anti-GPC4 sdAb, as defined in any one of claims 1-4; and - detecting the presence of the bound sdAb wherein the presence or expression level of GPC4 in the sample indicates that the subject is more likely to respond to treatment with the anti-cancer therapy; optionally wherein the anti-cancer treatment includes an anti-GPC4 sdAb as defined in any one of claims 1-4, a multivalent binding compound as defined in claims 5-6 or a chimeric antigen receptor as defined in claim 7.