CD33 specific single domain antibodies (SDAB), cars and other products derived therefrom, and their uses in therapy and diagnosis

Abstract

The present invention refers to anti-CD33 specific single domain antibodies, and their use in chimeric antigen receptor cells, mainly T cells, which are of use in the treatment of cancer characterized by an increase in cells expressing CD33, in particular acute myeloid leukemia.

Description

[0001] CD33 SPECIFIC SINGLE DOMAIN ANTIBODIES (sdAb), CARs AND OTHER PRODUCTS DERIVED THEREFROM, AND THEIR USES IN THERAPY AND DIAGNOSIS

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention belongs to the technical field of medicine and diagnosis, in particular to anti-CD33 specific single domain antibodies and uses thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Acute myeloid leukemia (AML) is a heterogeneous neoplasm characterized by uncontrolled clonal expansion of transformed immature myeloid hematopoietic precursors. It is the most common acute leukemia in adults with an incidence of approximately 4.2 cases per 100,000 habitants, and a median age of presentation of 68 years old, although it can be manifest at any age. Despite recent advances with the FDA approval of over 11 new therapies, prognosis in these patients particularly in the relapse and refractory setting is poor, with median overall survival bellow 8 months.

[0006] Chimeric antigen receptor T cells (CAR-T cells) are genetically engineered T cells designed to target specific epitopes. CAR-T cells against B cell antigens like CD 20, CD19 and BCMA have shown success in treating B cell malignancies, such as lymphoma, acute lymphoblastic leukemia, and multiple myeloma. However, CAR-T cells targeting non-B cell-associated epitopes have yet to achieve similar efficacy. Many efforts have been made to generate AML targeted immunotherapies, such as antibodydrug conjugates (ADCs), bispecific T cell engagers (BiTEs) and CAR-T cells, using myeloid markers such as CD123, CLL1 and CD33, or targeting immune-checkpoints like PD-1 ; CTLA4 and TIM-3. Most of these myeloid associated antigens are also expressed on myeloid stem precursors, so myelotoxicity is associated to these immunotherapies. For this reason, haematopoietic stem cell transplantation (HSCT) is required in all patients potentially treated with CAR-T cell therapy.

[0007] CD33 or Siglec-3 is a transmembrane receptor of the sialic acid-binding immunoglobulin- like lectin (SIGLEC) family and possesses an immunomodulating function. It is predominantly expressed on myeloid cells, including leukemic blasts in AML. The first CD33 targeted immunotherapy approved by the FDA was the ADC gemtuzumab- ozogamycin (GO) which demonstrated great therapeutic potential, nowadays being used in combined treatment for some CD33+ AML patients. CD33-targeted CAR-T therapies face several challenges such as i) on-target / off-tumor toxicity; ii) antigen escape; iii) Persistence and Exhaustion; and iv) Optimization of Dosing and Manufacturing.

[0008] In view of this, there is an urgent need for new therapeutic strategies for the treatment of AML, in particular strategies which target CD33.

[0009] SUMMARY OF THE INVENTION

[0010] The inventors have obtained a series of single domain antibodies (sdAb) which bind to human CD33. sdAb show several advantages over regular antibodies, such as being smaller, more stable, and having improved affinity and specificity for their target. These advantages make them ideally suited to be used as antigen receptors in CAR cells, providing an improved treatment for cancers characterized by an increase in CD33 expressing cells, in particular acute myeloid leukemia.

[0011] A first aspect of the present invention relates to a single domain antibody (sdAb) that specifically binds to human CD33, wherein:

[0012] (a) the CDR1 , CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 18, 19 and 20 or a functionally equivalent variant of one or more of the above;

[0013] (b) the CDR1 , CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 6, 7and 8 or a functionally equivalent variant of one or more of the above;

[0014] (c) the CDR1 , CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 9, 10 and 11 or a functionally equivalent variant of one or more of the above;

[0015] (d) the CDR1 , CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 12, 13 and 14 or a functionally equivalent variant of one or more of the above; or

[0016] (e) the CDR1 , CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 9, 13 and 17 or a functionally equivalent variant of one or more of the above.

[0017] A further aspect of the present invention relates to a biparatopic sdAb characterized in that it comprises a first and second sdAb wherein the first and / or second sdAb is a sdAb according to the invention. Another aspect of the present invention relates to a conjugate comprising:

[0018] (a) a first domain comprising the sdAb according to the invention or the biparatopic sdAb according to the invention, and

[0019] (b) a second active domain, preferably comprising a therapeutic, binding / targeting or marker agent.

[0020] Another aspect of the present invention relates to a polynucleotide encoding the sdAb or the biparatopic sdAb according to the invention or the conjugate according to the invention, wherein the conjugate is a fusion protein.

[0021] A further aspect of the present invention relates to a vector comprising the polynucleotide according to the invention.

[0022] Yet another aspect of the present invention relates to a host cell comprising the polynucleotide according to the invention or the vector according to the invention.

[0023] A further aspect of the present invention relates to a polynucleotide encoding for a CD33- specific chimeric antigen receptor (CAR) comprising:

[0024] (a) a sdAb according to the invention or the biparatopic sdAb according to the invention;

[0025] (b) a transmembrane domain; and

[0026] (c) at least one intracellular signaling domain and / or costimulatory domain.

[0027] Another aspect of the present invention relates to a vector comprising a polynucleotide encoding for a CD33-specific CAR according to the previous aspect.

[0028] A further aspect of the present invention relates to a cell comprising a polynucleotide encoding for a CD33-specific CAR or a vector comprising said polynucleotide as defined in the previous aspects.

[0029] One more aspect relates to an ex vivo method for obtaining a cell according to the invention comprising transducing the cell or precursor thereof with a polynucleotide according to the invention or with a vector comprising said polynucleotide, wherein the polynucleotide encodes for the sdAb according to the invention, the biparatopic sdAb according to the invention, the CD33-specific CAR according to the invention or for the conjugate according to the invention, whenever the conjugate is a fusion protein.

[0030] A further aspect relates to a pharmaceutical composition comprising the sdAb according to the invention, the biparatopic sdAb according to the invention, the conjugate according to the invention, the polynucleotide according to the invention, the vector according to the invention comprising said polynucleotide, and / or the cell according to the invention comprising said vector and / or said polynucleotide, wherein the polynucleotide encodes for the sdAb according to the invention, the biparatopic sdAb according to the invention, the CD33-specific CAR according to the invention or for the conjugate according to the invention, whenever the conjugate is a fusion protein.

[0031] Yet another aspect of the present invention relates to the sdAb according to the invention, the biparatopic sdAb according to the invention, the conjugate according to the invention, the polynucleotide according to the invention, the vector according to the invention comprising said polynucleotide, and / or the cell according to the invention comprising said vector and / or said polynucleotide for use in medicine, wherein the polynucleotide encodes for the sdAb according to the invention, the biparatopic sdAb according to the invention, the CD33-specific CAR according to the invention or for the conjugate according to the invention, whenever the conjugate is a fusion protein.

[0032] Another aspect of the present invention relates to the sdAb according to the invention, the biparatopic sdAb according to the invention, the conjugate according to the invention, the polynucleotide according to the invention, the vector according to the invention comprising said polynucleotide, and / or the cell according to the invention comprising said vector and / or said polynucleotide for use in the treatment of cancer, wherein the polynucleotide encodes for the sdAb according to the invention, the biparatopic sdAb according to the invention, the CD33-specific CAR according to the invention or for the conjugate according to the invention, whenever the conjugate is a fusion protein.

[0033] One more aspect relates to a method to detect the presence or level of CD33 in a sample comprising:

[0034] (a) contacting the sample with the sdAb according to the invention or the biparatopic sdAb according to the invention or the conjugate according to the invention so as to obtain a mixture wherein said sdAb, biparatopic sdAb or conjugate is allowed to substantially bind with said CD33 when the latter is present in said sample; and

[0035] (b) detecting the binding of the sdAb, of the biparatopic sdAb or of the conjugate to the CD33 present in the sample thereby providing an indication of the presence or of the level of CD33 in the sample. A final aspect relates to a method for the diagnosis of a disease characterized by an increased level of cells expressing CD33 in a patient, the method comprising detecting the level of cells expressing CD33 in a sample from the patient by a method as defined in any of claims of the invention wherein the presence of an increased number of cells expressing CD33 in the sample from the patient with respect to a reference value is indicative of the subject suffering from a disease characterized by an increased level of cells expressing CD33.

[0036] BRIEF DESCRIPTION OF THE FIGURES

[0037] Figure 1. Identification of novel sdAb against human CD33. (A) CD33 indirect ELISA curves of llama plasma at different stages during immunization (Percentage of signal vs plasma dilution factor (FD). (B) Output / input phage ratio on each panning round. (C) Monoclonal phage ELISA of the original 124 library and the final output OA3. (D) Preliminary ELISA screening of isolated E.coli clones infected with bacteriophages. As positive control, one positive clone prom a previous round output (OA2) was offered. (E) Final ELISA screening of isolated E.coli clones expressing the sdAbs from the expression vector pETMod. Positive control was C4 from the preliminary screening and negative control, an irrelevant sdAb expressing clone culture supernatant from a panning round against other target. (F) Phylogenetic tree of the identified sdAb sequences against CD33 according to half distance, clustered in 5 families. (G) Coomassie blue stained SDS-PAGE and anti-HA Western blot of the IMAC and IEC purified VHHs. (H) Cross reactivity test (ELISA) of the 5 candidate VHHs against antigens belonging to the 124 llama library.

[0038] Figure 2. sdAb binding characterization and activation profile of designed sdAb CARs. (A) ELISA sigmoidal curves for each candidate VHH (relative A450 vs log of VHH concentration (ng / mL) (B) Histograms of anti-HA PE stained MOLM13 CD33+ cells preincubated withl ug of each VHH for 1 h, and the control staining with antiCD33 BV510 (WM53) (C) SPR sensorgrams for each VHH from SCK runs, and their respective fitting curve. (D) BLI epitope binning curves of the secondary binding of each VHH, the first VHH to bind is explicated on each graph title. (E) Schematic representation of the second generation sdAb-based CAR construct design, the generation of lentiviral vectors and the final CAR expressed on T cells or Jurkat cells. (F) Histograms of EGFR staining representing CAR expression in Jurkat TPR cells, all of high percentage and similar distribution (CAR density). (G) Activation profile of CAR expressing Jurkat TPR cells. From left to right: NFAT (GFP reported), NFkB (BFP reported) and AP1 (mCherry reported) mediated activation of untreated cells (green bars) and co-cultured with MOLM13 in a 1 :1 ratio (light blue bars).

[0039] Figure 3. CAR-T cell phenotypic and functional characterization. (A) Population doublings during 14 days of CAR-T cell expansion. (B, C, D) Phenotypic characterization of CAR-T cells, and the untransduced control (UTD) on day 12-14 (final) and the initial T cells on day 0 before transduction (basal) be via flow cytometry (B) T cell subpopulations of CD4 (left) and CD8 (right) T cells, (C) CAR % and CD8 / CD4 populations, (D) Activation and exhaustion markers (top and bottom panels respectively) of CD4 (left) and CD8 (right) T cells. (E) CAR-T cytotoxicity on 3 different AML cell lines expressing different levels of CD33 (MFI of stained cells on left panel): MV411 , MOLM13 and HL60 evaluated via luciferase activity. (F) Cytokine expression in the supernatant of 1 :1 E:T co-cultures: IL2 (left) and IFNg (right). N = 8 independent healthy donors aged between 18-26 years old N = 4 for Nb1 (not included in the first experiments). Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparisons. * p< 0.05, ** p< 0.01 , *** p<0.001

[0040] Figure 4. CAR-T cell phenotypic and functional characterization after continued stimulation with tumoral cells. (A) Population doublings during 14 days of CAR-T cell continued stimulation. (B, C, D) Phenotypic characterization of CAR-T cells via flow cytometry (B) T cell subpopulations of CD4 (left) and CD8 (right) T cells, (C) CAR % and CD8 / CD4 populations, (D) Activation and exhaustion markers (top and bottom panels respectively) of CD4 (left) and CD8 (right) T cells. (E) CAR-T cytotoxicity on MOLM13 cells after continued stimulation. (F) Cytokine expression in the supernatant of 1 :1 E:T co-cultures: IL2 (left) and IFNg (right). N = 4 independent healthy donors aged between 18-26 years old N = 2 for Nb1 (not included in the first experiments). Statistical comparison between groups was analysed using two-way ANOVA with Tukey's multiple comparison. * p< 0.05, ** p< 0.01 , *** p<0.001

[0041] Figure 5. In vivo evaluation of CD33 targeted sdAb CAR-T cells on mouse xenograft AML model. (A) Schematic representation of the optimized procedure on NGS mice. (B, C, D) Kaplan-meyer survival curves of (B) low dose treatment (0.5 million CAR-T per mice) (C) high dose treatment (3 million CAR-T / mice) and (D) Intermediate dose treatment (1.5 million CAR-T / mice). N=10 for all groups. For statistical analysis survival curves were compared using the Log-rank (Mantel-Cox) test, ** p< 0.01 , *** p<0.001 (E) Luciferase activity measurements to evaluate tumoral progression in another in vivo experiment (dose 1.5 CAR-T / mice) N=4 (2 male, 2 female). Figure 6. ELISA CD33 binding assay for Nb16 and the CDR-grafted sdAb (Graft Nb16) using Nb16 natural CDRs and consensus framework regions derived from sdAb database.

[0042] Figure 7: In vivo evaluation of CAR-T cell expansion / persistence and phenotypes. A) Procedure details. B) Activation and exhaustion marker expression in CAR-T cells extracted from the spleen of sacrificed mice. C) Number of CAR-T cells over 100000 cells extracted from the spleen of treated mice on days 3 and 7 post tumor injection.

[0043] Figure 8: In vitro and in vivo evaluation of co-stimulatory CAR domains using Nb16- A) In vitro cytotoxicity and cytokine production. B) In vivo survival curves in mice AML xenograft model.

[0044] Figure 9: Specificity test over CD33- control cell line MM1S. A) Cytotoxicity test. B) Cytokine production.

[0045] Figure 10: Allogenic CAR-T cells based on Nb16. A) Optimized procedure description. B) Allogenic CAR-T cell characterization: TCR and HLA expression, CAR expansion and in vitro cytotoxicity

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] Single domain antibodies

[0048] A first aspect of the present invention relates to a single domain antibody (sdAb) that specifically binds to CD33 wherein: a) the CDR1 , CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 18, 19 and 20 or a functionally equivalent variant thereof b) the CDR1 , CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 6, 7 and 8 or a functionally equivalent variant thereof; c) the CDR1 , CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 9, 10 and 11 or a functionally equivalent variant thereof; d) the CDR1 , CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 12, 13 and 14 or a functionally equivalent variant thereof; or e) the CDR1 , CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 9, 13 and 17 or a functionally equivalent variant thereof.

[0049] As used herein the term “single domain antibody”, or its acronym “sdAb”, has its general meaning in the art and refers to the single heavy chain variable domain ( H or HH) from immunoglobulins. In one embodiment, these sdAbs derive from antibodies of the type that can be found in Camelid mammals (e.g. Lama glama), known as heavy chain-only antibodies (HcAbs), which are naturally devoid of light chains. Such single domain antibody is also known as “nanobody®”. In other embodiments sdAbs can derive form a natural or modified VH domain from conventional immunoglobulins, of human or other vertebrate species. In a particular embodiment, SdAbs can derive from VH domain present in new antigen recognition immunoglobulins (IgNAR) of cartilaginous fishes (e.g. Ginglymostoma cirratum), and such are known as vNAR. SdAb are characterized by a molecular weight of about 12-15 kDa, with a peptide chain of about 100-130 amino acids long, comprising three “complementarity-determining regions” (CDR), CDR1 , CDR2 and CDR3, which determine the antigen specificity and the binding properties of the sdAb. sdAbs can be obtain from several sources, natural and synthetic. Thus, in the meaning of the present invention, the term "single domain antibody" comprises polypeptides, which are derived from a non-human source, preferably a camelid, preferably a lama (Scientific name: Llama glama) heavy chain-only antibody, obtained by recombinant DNA technology from natural HcAb coding mRNA extracted from B cells. SdAbs may be humanized by grafting the CDRs into human or humanized framework regions (FR) further described. In a preferred embodiment the single domain antibody (sdAb) that specifically binds to CD33 are humanized. Moreover, the term comprises polypeptides derived from non-camelid sources, e.g. mouse or human, which have been "camelized", as previously described, e.g. in WO 08 / 101985 and WO 08 / 142164. The term "single domain antibody" encompasses immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. It also includes fully human, humanized or chimeric immunoglobulin sequences. For example, it comprises camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized single variable domains.

[0050] As used herein, the term "CDR" refers to the complementarity-determining region within sdAb variable sequences and corresponds to a sdAb region having a structure that is complimentary to its target antigen or epitope. Other portions of the sdAb, not interacting with antigen, are referred to as "framework regions" (FRs). As previously mentioned, the sdAb comprises regions of hypervariability, also known as "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, which are known as "framework regions" ("FR"). sdAb have four framework regions (FRs) each positioned before, after, and between CDR regions. sdAb framework regions are referred to herein as FR1 , FR2, FR3, and FR4. FRs and CDRs of sdAb are typically in the order of FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4, from N- to C-terminus. In a preferred embodiment of the sdAb according to the previous aspect:

[0051] (a) the FR1 , FR2, FR3 and FR4 regions in the sdAb as defined above under

[0052] (a) of the first aspect of the invention comprise respectively the sequences of SEQ ID NO: 33, 34, 35 and 25 or a functionally equivalent variant of one or more of the above;

[0053] (b) the FR1 , FR2, FR3 and FR4 regions in the sdAb as defined above under

[0054] (b) of the first aspect of the invention comprise respectively the sequences of SEQ ID NO: 22, 23, 24 and 25 or a functionally equivalent variants of one or more of the above;

[0055] (c) the FR1 , FR2, FR3 and FR4 regions in the sdAb as defined above under

[0056] (c) of the first aspect of the invention comprise respectively the sequences of SEQ ID NO: 26, 23, 27 and 25 or a functionally equivalent variant of one or more of the above;

[0057] (d) the FR1 , FR2, FR3 and FR4 regions in the sdAb as defined above under

[0058] (d) of the first aspect of the invention comprise respectively the sequences of SEQ ID NO: 28, 29, 30 and 25 or a functionally equivalent variant of one or more of the above; or

[0059] (e) the FR1 , FR2, FR3 and FR4 regions in the sdAb as defined above under

[0060] (e) of the first aspect of the invention comprise respectively the sequences of SEQ ID NO: 31 , 23, 32 and 25 or a functionally equivalent variant of one or more of the above.

[0061] The extent of the framework region and CDRs can be precisely identified using methodologies known in the art, for example, by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or (e.g., and) the contact definition, all of which are well known in the art. As used herein, a CDR may refer to the CDR defined by any method known in the art. Two sdAb having the same CDR means that the two antibodies have the same amino acid sequence of that CDR as determined by the same method, for example, the IMGT definition.

[0062] As defined above, the invention relates to an sdAb which is defined by the presence of certain specific CDRs and FR but which encompasses also sdAb which contain a functionally equivalent variants of one or more of the CDR and functionally equivalent variants of one or more of the FRs.

[0063] The expression “functionally equivalent variant” when referred to a CDR refers to a peptide sequence which, when present in a sdAb in combination with the other CDRs of the sdAb, results in an sdAb which substantially preserves its ability of specifically binding to the antigen.

[0064] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds," refer to sdAb binding to an epitope on a predetermined antigen with high affinity.

[0065] As used herein, the term "high affinity" for an sdAb refers to an sdAb having a Ko of 10'7M or less, preferably 10'8M or less, more preferably 10'9M or less and even more preferably 10'1° M or less for a target antigen. Typically, the sdAb (i) binds with an equilibrium dissociation constant (KD) of approximately less than 10'7M, such as approximately less than 10'8M, 10'9M or 10'1° M or even lower when determined by, e.g., surface plasmon resonance (SPR) technology in a BIACORE instrument using the predetermined antigen, e.g., CD33, as the analyte and the sdAb as the ligand, or Scatchard analysis of binding of the antibody to antigen positive cells, and (ii) binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. Accordingly, a sdAb that "specifically binds to CD33" refers to a sdAb that binds to soluble or cell bound human CD33 with a Ko of 10'7M or less, such as approximately less than 10'8M, 10'9M or 10'1° M or even lower.

[0066] The functionality of the sdAb carrying the functionally equivalent variant of the CDRs according to the invention can also be determined by measuring their “half maximum effective concentration” (ECso), which is refers to the half-maximal effect concentration (concentration for 50% of maximal effect, ECso), which refers to the concentration that elicits 50% of the maximal effect. In a preferred embodiment the sdAb according to the invention have an ECso of at least 1 nM, at least 2 nM, at least 3 nM, at least 4 nM, at least 5 nM, at least 6 nM, at least 7 nM, at least 8 nM, at least 9 nM, at least 10 nM, at least 10 nM. at least 15 nM, at least 20 nM, at least 25 nM, at least 30 nM, at least 35 nM, at least 40 nM, at least 45 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 120 nM, at least 140 nM, at least 160 nM, at least 180 nM, at least 200 nM. It will be understood that the functionally equivalent variants of the sdAbs will maintain at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% of the EC50 of the sdAb to which the variant refers to. Suitable functionally equivalent variants of the CDRs of the sdAbs according to the present invention include those which, if n is the total number of amino acids of the CDR, contain amino acid substitution at n-1 , n-2, n-3, n-4, n-5 and so forth positions of the reference CDR. For instance, if the reference CDR consists of 10 residues, then the functionally equivalent variant of the CDR contains 9, 8, 7 6, 5, 4, 3, 2 or 1 substitutions. The substitutions can be of conservative nature.

[0067] "Conservative amino acid substitutions" result from replacing one amino acid with another having similar structural and / or chemical properties. For example, the following six groups each contain amino acids that are conservative substitutions for one another:

[0068] 1) Alanine (A), Serine (S), Threonine (T);

[0069] 2) Aspartic acid (D), Glutamic acid (E);

[0070] 3) Asparagine (N), Glutamine (Q);

[0071] 4) Arginine (R), Lysine (K);

[0072] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0073] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0074] In a preferred embodiment of the invention the sdAb according to the invention wherein the functionally equivalent variant of SEQ ID NO: 20 is SEQ ID NO: 21.

[0075] In a preferred embodiment of the invention the sdAb contains the CDR1 , CDR2 and CDR3 according to the sequences SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 21 , respectively.

[0076] The expression “functionally equivalent variant”, when referred to a FR refers to a peptide sequence which, when present in a sdAb in combination with the other FRs of the sdAb, results in an sdAb which substantially preserves its ability of specifically binding to the antigen. The term "specific binding," as well as suitable values for the parameters used to measure binding (KD and EC50) have been defined above in the context of the functionally equivalent variant of the CDRs.

[0077] Suitable functionally equivalent variants of the FRs of the sdAbs according to the present invention include those which, if n if the total number of amino acids of the FR, contain amino acid substitution at n-1 , n-2, n-3, n-4, n-5 and so forth positions of the reference CDR. For instance, if the reference FR consists of 10 residues, then the functionally equivalent variant of the FR contains 9, 8, 7, 6, 5, 4, 3, 2 or 1 substitutions. The substitutions can be of conservative nature. The term “conservative amino acid substitutions" has been defined above in the context of the functionally equivalent variants of the CDRs and applies equally in the case of the functionally equivalent variant of the FRs.

[0078] In another preferred embodiment of the invention the sdAb according to the invention wherein the functionally equivalent variant of SEQ ID NO: 22, 26, 28, 31 or 33 is SEQ ID NO: 62.

[0079] In another preferred embodiment of the invention the sdAb according to the invention wherein the functionally equivalent variant of SEQ ID NO: 23, 29, 34 or 36 is SEQ ID NO: 63.

[0080] In another preferred embodiment of the invention the sdAb according to the invention wherein the functionally equivalent variant of SEQ ID NO: 34 is SEQ ID NO: 36 or SEQ ID NO: 63.

[0081] In another preferred embodiment the sdAb according to the invention characterized in that:

[0082] (a) it comprises a sequence according to SEQ ID NO: 5 or a functionally equivalent variant thereof;

[0083] (b) it comprises a sequence according to SEQ ID NO: 1 or a functionally equivalent variant thereof;

[0084] (c) it comprises a sequence according to SEQ ID NO: 2 or a functionally equivalent variant thereof;

[0085] (d) it comprises a sequence according to SEQ ID NO: 3 or a functionally equivalent variant thereof; or

[0086] (e) it comprises a sequence according to SEQ ID NO: 4 or functionally equivalent variant thereof.

[0087] The expression “functionally equivalent variant”, when referred to a certain sdAb refers to a polypeptide resulting from the modification, deletion or insertion or one or more amino acids and which substantially preserves the activity of its reference polypeptide wherein, if the variant results from the modification of one or more residues, then the modification can be a conservative amino acid substitution or not.

[0088] The term "specific binding," as well as values for the parameters used to measure binding (KD and EC50) have been defined above in the context of the functionally equivalent variant of the CDRs. In a preferred embodiment the sdAb according to the invention wherein:

[0089] (a) the functionally equivalent variant of SEQ ID NO: 1 comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% identity with SEQ ID NO: 1 ;

[0090] (b) the functionally equivalent variant of SEQ ID NO: 2 comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% identity with SEQ ID NO: 2;

[0091] (c) the functionally equivalent variant of SEQ ID NO: 3 comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% identity with SEQ ID NO: 3;

[0092] (d) the functionally equivalent variant of SEQ ID NO: 4 comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% identity with SEQ ID NO: 4; or

[0093] (e) the functionally equivalent variant of SEQ ID NO: 5 comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% identity with SEQ ID NO: 5.

[0094] The degree of identity or similarity between two polypeptides or two polynucleotides is determined by using computer-implemented algorithms and methods that are widely known in the art. The identity and similarity between two sequences of amino acids is preferably determined using the BLASTP algorithm or determined using the Needleman- Wunsch algorithm to obtain an optimal alignment between two sequences. This analysis can be performed using an online software such as EMBOSS Needle (https: / / www.ebi.ac.uk / jdispatcher / psa / emboss_needle). In a preferred embodiment of the invention the degree of identity between two polypeptides is determined across the whole length of both polypeptides.

[0095] It will be understood that the functionally equivalent variants of the sdAbs as defined in any of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 may result from the modification, deletion or insertion of one or more amino acids at one or more of the FR regions, from the modification, deletion or insertion of one or more amino acids at one or more of the CDR or from the modification, deletion or insertion of one or more amino acids at one or more of the FRs and of the CDRs.

[0096] In some embodiments, the functionally equivalent variant of the sdAbs as defined in any of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 results from the modification, deletion or insertion of one or more amino acids in the CDR1 , while the CDR2 and CDR3 remain unaltered with respect to the sdAb. In some embodiments, if n is the total number of amino acids of the CDR1 of the sdAb, the functionally equivalent variant results from the modification of 1 , 2, 3, and up to n-1 positions in said CDR1 , wherein said modifications are preferably conservative substitutions.

[0097] In some embodiments, the functionally equivalent variant of the sdAbs as defined in any of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 results from the modification, deletion or insertion of one or more amino acids in the CDR2, while the CDR1 and CDR3 remain unaltered with respect to the sdAb. In some embodiments, if n is the total number of amino acids of the CDR2 of the sdAb, the functionally equivalent variant results from the modification of n-1 , n-2, n-3, n-4, n-5 and so forth positions in said CDR2.

[0098] In some embodiments, the functionally equivalent variant of the sdAbs as defined in any of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 results from the modification, deletion or insertion of one or more amino acids in the CDR3 region, while the CDR1 and CDR2 remain unaltered with respect to the sdAb. In some embodiments, if n is the total number of amino acids of the CDR3 of the sdAb, the functionally equivalent variant results from the modification of 1 , 2, 3, and up to n-1 positions in said CDR3, wherein said modifications are preferably conservative substitutions.

[0099] In some embodiments, the functionally equivalent variant of the sdAbs as defined in any of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 results from the modification, deletion or insertion of one or more amino acids in the CDR1 , and CDR2, while the CDR3 remains unaltered with respect to the sdAb. In some embodiments, if n is the total number of amino acids of the CDR1 and CDR2 of the sdAb, the functionally equivalent variant results from the modification of 1 , 2, 3 and up to n-1 positions in said CDR1 and CDR2, wherein said modifications are preferably conservative substitutions. In some embodiments, the functionally equivalent variant of the sdAbs as defined in any of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 results from the modification, deletion or insertion of one or more amino acids in the CDR1 and CDR3, while the CDR2 remains unaltered with respect to the sdAb. In some embodiments, if n is the total number of amino acids of the CDR1 and CDR3 of the sdAb, the functionally equivalent variant results from the modification of 1 , 2, 3 and up to n-1 positions in said CDR1 and CDR3, wherein said modifications are preferably conservative substitutions.

[0100] In some embodiments, the functionally equivalent variant of the sdAbs as defined in any of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 results from the modification, deletion or insertion of one or more amino acids in the CDR2 and CDR3, while the CDR1 remains unaltered with respect to the sdAb. In some embodiments, if n is the total number of amino acids of the CDR2 and CDR3 of the sdAb, the functionally equivalent variant results from the modification of 1 , 2, 3, and up to n-1 positions in said CDR2 and CDR3, wherein said modifications are preferably conservative substitutions.

[0101] In some embodiments, the functionally equivalent variant of the sdAbs as defined in any of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 results from the modification, deletion or insertion of one or more amino acids in the CDR1 , in the CDR2 and in the CDR3. In some embodiments. In some embodiments, if n is the total number of amino acids of the CDR1 , CDR2 or CDR3 of the sdAb, the functionally equivalent variant results from the modification of 1 , 2, 3, and up to n-1 positions in said CDR1 , CDR2 and CDR3, wherein said modifications are preferably conservative substitutions.

[0102] In some embodiments, the functionally equivalent variant of the sdAbs as defined in any of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 results from the modification, deletion or insertion of one or more amino acids in the FR1 , while the FR2, FR3 and FR4 remain unaltered with respect to the sdAb. In some embodiments, if n is the total number of amino acids of the FR1 of the sdAb, the functionally equivalent variant results from the modification of 1 , 2, 3 and up to n-1 positions in said FR1 , wherein said modifications are preferably conservative substitutions.

[0103] In some embodiments, the functionally equivalent variant of the sdAbs as defined in any of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 results from the modification, deletion or insertion of one or more amino acids in the FR2, while the FR1 , FR3 and FR4 remain unaltered with respect to the sdAb. In some embodiments, if n is the total number of amino acids of the FR2 of the sdAb, the functionally equivalent variant results from the modification of 1 , 2, 3 and up to n-1 positions in said FR2, wherein said modifications are preferably conservative substitutions.

[0104] In some embodiments, the functionally equivalent variant of the sdAbs as defined in any of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 results from the modification, deletion or insertion of one or more amino acids in the FR3, while the FR1 , FR2 and FR4 remain unaltered with respect to the sdAb. In some embodiments, if n is the total number of amino acids of the FR3 of the sdAb, the functionally equivalent variant results from the modification of 1 , 2, 3 and up to n-1 positions in said FR3, wherein said modifications are preferably conservative substitutions.

[0105] In some embodiments, the functionally equivalent variant of the sdAbs as defined in any of SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 results from the modification, deletion or insertion of one or more amino acids in the FR4, while the FR1 , FR2 and FR3 remain unaltered with respect to the sdAb. In some embodiments, if n is the total number of amino acids of the FR4 of the sdAb, the functionally equivalent variant results from the modification of 1 , 2, 3 and up to n-1 positions in said FR4, wherein said modifications are preferably conservative substitutions.

[0106] In a preferred embodiment of the invention the sdAb according to the invention wherein the functionally equivalent variant of SEQ ID NO: 5 is one which comprises, consists or consists essentially of SEQ ID NO: 37 or of SEQ ID NO: 64.

[0107] In some embodiments, the sdAbs according to the present invention are defined by the CDR and FRs as shown in the following table:

[0108] The sdAbs of the present invention, and functionally equivalent variants thereof, have a high affinity for the human CD33 or Siglec-3. The term “CD33”, also known as “Siglec-3” (sialic acid binding Ig-like lectin 3, SIGLEC3, SIGLEC-3) “gp67” or “p67”, as known herein refers to a transmembrane receptor expressed on cells of myeloid lineage. It is usually considered myeloid-specific, but it can also be found on some lymphoid cells. CD33 binds sialic acids, being a member of the SIGLEC family of lectins. CD33 contains two immunoglobulin domains (one IgV and one lgC2 domain) in the extracellular portion and an immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the intracellular portion. CD33 can be stimulated by any molecule with sialic acid residues such as glycoproteins or glycolipids. Upon binding, the immunoreceptor tyrosine-based inhibition motif (ITIM) of CD33, present on the cytosolic portion of the protein, is phosphorylated and acts as a docking site for Src homology 2 (SH2) domaincontaining proteins like SHP phosphatases. This results in a cascade that inhibits phagocytosis in the cell.

[0109] CD33 is associated with Alzheimer’s disease since its control of microglial activation gets altered in the presence of amyloid and Tau proteins. Furthermore, CD33 is also associated with acute myeloid leukemia (AML) as CD33 expression levels have been shown to correlate with molecular aberrations in AML.

[0110] The term “Alzheimer’s” as used herein refers to a progressive neurodegenerative disease characterized by memory and cognitive dysfunction. It mostly occurs in the elderly with a slow and irreversible course. According to the development of cognitive dysfunction, Alzheimer's disease can be divided into early, middle and late stages. In the early stage, patients have no obvious symptoms, most of them only show forgetfulness and anxiety, so it is difficult to detect or diagnose; in the middle stage, there will be chaotic symptoms, personality and spleen will change, and memory will also appear confused; in the later stage, the patients will be completely Dementia, without the ability to take care of oneself, will eventually die from multiple diseases.

[0111] The term “acute myeloid leukemia”, or its acronym “AML”, as used herein refers to a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. Acute myeloid leukemia (AML) is a genetically heterogeneous disease, with a highly variable prognosis and an overall high mortality rate. The 5-year overall survival of adult AML patients is less than 50%, and only 20% of elderly patients survive over 2 years.

[0112] The term “cells of the myeloid lineage” as used herein refers to cells which develop during the process of myelopoiesis and include Granulocytes, Monocytes, Megakaryocytes, and Dendritic Cells. Circulating Erythrocytes and Platelets also develop from myeloid progenitor cells. Many of these myeloid cells are short-lived, and immune system homeostasis requires continual HSC self-renewal and differentiation.

[0113] Biparatopic sdAb

[0114] The single domain antibodies herein disclosed can also be used to obtain biparatopic sdAb antibodies.

[0115] As used herein, the term “biparatopic” sdAb means a sdAb comprising a single domain antibody and a second single domain antibody as herein defined, wherein these two single domain antibodies are capable of binding to two different epitopes of one antigen (e.g. BMCA), which epitopes are not normally bound at the same time by one monospecific immunoglobulin, such as e.g. a conventional antibody or one single domain antibody.

[0116] The biparatopic antibodies according to the present invention are characterized in that they comprise a first and second sdAb wherein the first and / or the second sdAb are sdAbs according to the invention.

[0117] All previous aspects and embodiments described previously in relation to the sdAbs are equally applicable to the biparatopic sdAbs.

[0118] It will be understood by the expert in the field that the biparatopic sdAb will maintain the binding affinity or show improved binding affinity with respect to that of the individual sdAb which compose the biparatopic sdAb.

[0119] As the expert in the field is aware that a biparatopic sdAb comprises two sdAbs polypeptides which are fused together. As such, the biparatopic polypeptide can be organized in two configurations. In a preferred embodiment of the invention the biparatopic sdAb according to the invention wherein the first sdAb and the second sdAb are arranged such that the first sdAb is N-terminally located in relation to the second sdAb or such that the first sdAb is C-terminally located in relation to the second sdAb.

[0120] The terms “N-terminally” and “C-terminally”, also referring to “N-terminal” and “C- terminal”, do not mean that the components need to be directly conjugated end-to-end, but that they maintain that relative order of positions regardless of the presence of additional elements at the end of either component or intercalated between them. The two sdAbs of the biparatopic sdAb can be fused end to end or may include one or more optional peptide or polypeptide “linkers” or “spacers” between them, linked preferably by a peptide bond. In a preferred embodiment of the invention the biparatopic sdAb according to the invention further comprising a peptide linker between the first and the second sdAb.

[0121] The effect of the linker region is to provide space between the two sdAbs without hindrance to their function of antigen binding. It is thus assured that the secondary and tertiary structure of one sdAb is not affected by the presence of either of the others. The spacer is of a polypeptide nature. The linker peptide preferably comprises at least 2 amino acids, at least 3 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids or approximately 100 amino acids.

[0122] The spacer or linker can be bound to the two sdAb of the biparatopic sdAb of the invention by means of covalent bonds, preferably by peptide bonds; and also preferably the spacer is essentially afunctional, and / or is not prone to proteolytic cleavage, and / or does not comprise any cysteine residue. Similarly, the three-dimensional structure of the spacer is preferably linear or substantially linear.

[0123] Preferred examples of linker peptides comprise 2 or more amino acids selected from the group consisting of glycine, serine, alanine and threonine. A preferred example of a flexible linker is a polyglycine linker. The possible examples of linker / spacer sequences include (GS)n, (GSGGS)n (SEQ ID NO: 52) and (GGGGS)n (SEQ ID NO: 53), where n is an integer of at least one, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used as both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Other examples of linkers are the sequences GGGGSGGGGSGGGGS (SEQ ID NO: 54) or GGSGGAP (SEQ ID NO: 55) or GGGVEGGG (SEQ ID NO: 60). In a preferred embodiment of the invention, the flexible peptide linker comprises a sequence according to SEQ ID NO: 53 (GGGGS) or SEQ ID NO: 54 (GGGGSGGGGSGGGGS).

[0124] Conjugate The sdAbs according to the invention can also be used to obtained conjugates wherein other domains of interest are fused or conjugated to the sdAbs. Another aspect of the present invention relates to a conjugate comprising:

[0125] (a) a first domain comprising the sdAb according to the invention or the biparatopic sdAb according to the invention, and

[0126] (b) a second active domain; preferably, comprising a therapeutic, binding / targeting or marker agent.

[0127] The term “conjugate”, as used herein, refers to any compound resulting from the attachment of two or more individual compounds, and more particularly to a covalent attachment of said individual compounds.

[0128] In an embodiment of the present invention, the conjugate comprises the sdAb or biparatopic sdAb region and at least one therapeutic, binding / targeting or marker agent which are covalently coupled, being said coupling direct or via a linking compound.

[0129] The terms "covalent coupling” or “covalent attachment" mean that the sdAb or conjugate and at least one therapeutic, binding / targeting or marker agent are either directly covalently joined through a chemical covalent bond to one another, or else are indirectly covalently joined to one another through an intervening moiety or moieties, such as a linker, or a bridge, or a spacer, moiety or moieties.

[0130] The term “active,” as used herein, refers to a portion, domain or fragment of an agent such as a molecule, compound, or polypeptide, having a biological activity or biological function.

[0131] The second active domain of the conjugate refers to an agent, in particular to a therapeutic agent, a binding or targeting agent, and / or a marker agent.

[0132] The number of therapeutic, binding / targeting or marker agents which are conjugated to the sdAb or the biparatopic sdAb, while not being particularly limitative, will depend on the number of available residues in the sdAbs polypeptide(s) which are available for chemical conjugation with the therapeutic, binding / targeting or marker agent. Since most conjugations occur via amino- or sulfhydryl groups present in the side chains of the amino acids forming part of the sdAbs polypeptide (s), the number of therapeutic, binding / targeting or marker agents conjugated to the sdAbs polypeptides will depend on the number of lysine and arginine residues (for a conjugation via an amino groups in the side chains) or on the number of cysteine residues (for conjugation via sulphidryl groups in the side chains) as well as on the yield of the conjugation reaction. Thus, in a particular embodiment of the invention, the conjugate according to the invention is conjugated to at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30 therapeutic, binding / targeting or marker agents.

[0133] In the cases wherein the conjugate is obtained with the present of an intervening moiety or moieties which mediates the bond between the first domain and the therapeutic, binding / targeting or marker agent, said intervening moiety is a bifunctional cross-linker and, more preferably, a heterobifunctional cross-linker, that reacts with the groups in the therapeutic, binding / targeting or marker agent and in the polypeptides of the first domain of the conjugate, either sequentially (either reacting with the therapeutic, binding / targeting or marker agent first and then with the polypeptides of the first domain of the conjugate, or first with the polypeptides of the first domain of the conjugate and then with the therapeutic, binding / targeting or marker agent) or simultaneously, using among other linkages such as thioethers, amide bonds, carbon-nitrogen double bonds, or linkages generated by cycloaddition. As a way of example typical thiol-reactive functional groups include iodoacetamides, maleimides, and disulfides. In addition, a sdAb can be treated with a small molecule or surface displaying an activated ester (e.g., an N-hydroxysuccinimidyl ester) to form amide bonds with the amino groups on lysine side chains and the N terminus. In an embodiment of the conjugate according to the invention, the intervening moiety is a heterobifunctional cross-linker which contains reactive groups capable of reacting with a thiol group and with an amino group. In one embodiment, the heterobifunctional cross-linker is 6-maleimidohexanoic acid N- hydroxysuccinimide ester.

[0134] In a preferred embodiment the conjugate is a fusion protein in which the therapeutic, binding / targeting or marker agent is a polypeptide fused with the sdAb or biparatopic sdAb.

[0135] The term “fusion protein” is well known in the art, referring to a single polypeptide chain artificially designed which comprises two or more sequences from different origins, natural and / or artificial. The fusion protein, per definition, is never found in nature as such.

[0136] The term “peptide” or “polypeptide”, as used herein, generally refers to a linear chain of around 2 to 40 amino acid residues joined together with peptide bonds. It will be understood that the terms “peptide bond”, “peptide”, “polypeptide” and protein are known to the person skilled in the art. From here on, “peptide” and “polypeptide” will be used indistinctly. As used herein, an "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative or any amino acid mimic known in the art. In certain embodiments, the residues of the protein or peptide are sequential, without any non-amino acid interrupting the sequence of amino acid residues. In other embodiments, the sequence may comprise one or more non-amino acid moieties. In particular embodiments, the sequence of residues of the protein or peptide may be interrupted by one or more non- amino acid moieties.

[0137] As previously mentioned, the arrangement in which the sdAb or biparatopic sdAb and the therapeutic, binding / targeting or marker agent are fused together in the fusion protein can differ. As such, in a preferred embodiment of the invention, the first domain and the second domain of the fusion protein are arranged such that the first domain is N- terminally located in relation to the second domain or such that the first domain is C- terminally located in relation to the domain region.

[0138] The term "therapeutic", as used herein in relation to the therapeutic agents, is used in a generic sense and includes treating agents, prophylactic agents, and replacement agents.

[0139] The term “therapeutic agent”, as used herein, is drawn to any compound, without chemical structure limitations, suitable for therapy and / or treatment of a condition, disorder or disease.

[0140] The nature of the therapeutic agent is not particularly limiting for the present invention provided it remains active in the conjugate or can be activated once it is delivered to the target cells, which are those which possess CD33 in the surface, i.e., cell of myeloid lineage. Accordingly, any therapeutic agent can be used in the conjugate provided that it shows an activity or can reach an activity once it reaches the CD33 target of at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50% or less of the activity of the unconjugated therapeutic agent. Alternatively, since the purpose of the first domain of the conjugate is to facilitate the action of the therapeutic agent by increasing its selectivity and reducing its off-target effects, it is contemplated that the effects of the therapeutic agent of the conjugate may be synergistic and exceed the parametrized values already known for the specific therapeutic agent. Accordingly, it is intended that some embodiments of the therapeutic agent bound to the conjugate according to the invention also show at least 101 %, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 175%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000%, or more of the functionality of the therapeutic agent alone.

[0141] In a preferred embodiment of the invention the therapeutic agent is a therapeutic polypeptide.

[0142] The term “therapeutic polypeptide”, as used herein, is drawn to any polypeptide suitable for therapy and / or treatment of a condition, disorder or disease, preferably the treatment of cancer, more preferably the treatment of leukemia, acute myeloid leukemia, lymphoma or multiple myeloma, more preferably the treatment of acute myeloid leukemia.

[0143] In an embodiment of the invention, the therapeutic agent is selected from the group consisting of

[0144] (i) a chemotherapy agent,

[0145] (ii) a cytotoxic polypeptide,

[0146] (iii) an antiangiogenic polypeptide,

[0147] (iv) a polypeptide encoded by a tumor suppressor gene,

[0148] (v) pro-apoptotic polypeptide,

[0149] (vi) a polypeptide having anti-cancer or anti-metastatic activity,

[0150] (vii) a polypeptide encoded by a polynucleotide which is capable of activating the immune response towards a tumor,

[0151] (viii) an antiangiogenic molecule, and

[0152] (ix) a toxin.

[0153] It will be understood that the term “chemotherapeutic agents” refers to anti-cancer agents.

[0154] As used herein, an anti-cancer agent is an agent that at least partially inhibits the development or progression of a cancer, including inhibiting in whole or in part symptoms associated with the cancer even if only for the short term.

[0155] Several anti-cancer agents can be categorized as DNA damaging agents and these include topoisomerase inhibitors (e.g., etoposide, ramptothecin, topotecan, teniposide, mitoxantrone), DNA alkylating agents (e.g., cisplatin, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chorambucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), DNA strand break inducing agents (e.g., bleomycin, doxorubicin, daunorubicin, idarubicin, mitomycin C), anti-microtubule agents (e.g., vincristine, vinblastine), anti-metabolic agents (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin, chlorodeoxyadenosine), anthracyclines, vinca alkaloids, or epipodophyllotoxins.

[0156] Additional examples of anti-cancer agents include without limitation Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Bortezomib (VELCADE); Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin (a platinum- containing regimen); Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin (a platinum-containing regimen); Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin; Decitabine; Dexormaplatin; Dezaguanine; Diaziquone; Docetaxel (TAXOTERE); Doxorubicin; Droloxifene; Dromostanolone; Duazomycin; Edatrexate; Eflornithine; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin; Erbulozole; Erlotinib (TARCEVA), Esorubicin; Estramustine; Etanidazole; Etoposide; Etoprine; Fadrozole; Fazarabine; Fenretinide; Floxuridine; Fludarabine; 5-Fluorouracil; Flurocitabine; Fosquidone; Fostriecin; Gefitinib (IRESSA), Gemcitabine; Hydroxyurea; Idarubicin; Ifosfamide; llmofosine; Imatinib mesylate (GLEEVAC); Interferon alpha-2a; Interferon alpha-2b; Interferon alpha-nl; Interferon alpha-n3; Interferon beta-l a; Interferon gamma-l b; Iproplatin; Irinotecan; Lanreotide; Lenalidomide (REVLLMID, REVIMID); Letrozole; Leuprolide; Liarozole; Lometrexol; Lomustine; Losoxantrone; Masoprocol; Maytansine; Mechlorethamine; Megestrol; Melengestrol; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pemetrexed (ALIMTA), Pegaspargase; Peliomycin; Pentamustine; Pentomone; Peplomycin; Perfosfamide; Pipobroman; Piposulfan; Piritrexim Isethionate; Piroxantrone; Plicamycin; Plomestane; Porfimer; Porfiromycin; Prednimustine; Procarbazine; Puromycin; Pyrazofurin; Riboprine; Rogletimide; Safingol; Semustine; Simtrazene; Sitogluside; Sparfosate; Sparsomycin; Spirogermanium; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tamsulosin; Taxol; Taxotere; Tecogalan; Tegafur; Teloxantrone; Temoporfin; Temozolomide (TEMODAR); Teniposide; Teroxirone; Testolactone; Thalidomide (THALOMID) and derivatives thereof; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan; Toremifene; Trestolone; Triciribine; Trimetrexate; Triptorelin; Tubulozole; Uracil; Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine; Vincristine; Vindesine; Vinepidine; Vinglycinate; Vinleurosine; Vinorelbine; Vinrosidine; Vinzolidine; Vorozole; Zeniplatin; Zinostatin; Zorubicin.

[0157] The anti-cancer agent may be an enzyme inhibitor including without limitation tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, or an EGFR inhibitor. The tyrosine kinase inhibitor may be without limitation Genistein (4', 5, 7- trihydroxyisoflavone), Tyrphostin 25 (3,4,5-trihydroxyphenyl), methylene]- propanedinitrile, Herbimycin A, Daidzein (4',7-dihydroxyisoflavone), AG-126, trans-1- (3'- carboxy-4'-hydroxyphenyl)-2-(2",5"-dihydroxy-phenyl)ethane, or HDBA (2- Hydroxy5- (2,5-Dihydroxybenzylamino)-2-hydroxybenzoic acid. The CDK inhibitor may be without limitation p21 , p27, p57, pl5, pl6, pl8, or pl9. The MAP kinase inhibitor may be without limitation KY12420 (C23H24O8), CNI-1493, PD98059, or 4-(4- Fluorophenyl)-2-(4- methylsulfinyl phenyl)-5-(4-pyridyl) IH-imidazole. The EGFR inhibitor may be without limitation erlotinib (TARCEVA), gefitinib (IRESSA), WHI- P97 (quinazoline derivative), LFM-A12 (leflunomide metabolite analog), ABX-EGF, lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788, and AG1458.

[0158] The anti-cancer agent may be a VEGF inhibitor including without limitation bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorin. The anti-cancer agent may be an antibody or an antibody fragment including without limitation an antibody or an antibody fragment including but not limited to bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), alemtuzumab (CAMPATH, indicated for B cell chronic lymphocytic leukemia,), gemtuzumab (MYLOTARG, hP67.6, anti-CD33, indicated for leukemia such as acute myeloid leukemia), rituximab (RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for B cell malignancy), MDX-210 (bispecific antibody that binds simultaneously to HER-2 / neu oncogene protein product and type I Fc receptors for immunoglobulin G (IgG) (Fc gamma Rl)), oregovomab (OVAREX, indicated for ovarian cancer), edrecolomab (PANOREX), daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated for respiratory conditions such as RSV infection), ibritumomab tiuxetan (ZEVALIN, indicated for Non-Hodgkin's lymphoma), cetuximab (ERBITUX), MDX-447, MDX-22, MDX-220 (anti-TAG-72), I0R- 05, 10R-T6 (anti-CD 1), IOR EGF / R3, celogovab (ONCOSCINT OV 103), epratuzumab (LYMPHOCIDE), pemtumomab (THERAGYN), and Gliomab-H (indicated for brain cancer, melanoma).

[0159] It is contemplated that in certain embodiments of the invention a protein that acts as an angiogenesis inhibitor is targeted to a tumor. These agents include, in addition to the anti-angiogenic polypeptides mentioned above, Marimastat; AG3340; COL-3, BMS- 275291 , Thalidomide, Endostatin, SLI5416, SLI6668, EMD121974, 2-methoxyoestradiol, carboxiamidotriazole, CMIOI, pentosan polysulphate, angiopoietin 2 (Regeneron), herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM- 1470, platelet factor 4 or minocycline.

[0160] Other suitable active agents are DNA cleaving agents. Examples of DNA cleaving agents suitable for inclusion as a cell toxin in the conjugates used in practicing the methods include, but are not limited to, anthraquinone-oligopyrrol-carboxamide, benzimidazole, leinamycin; dynemycin A; enediyne; as well as biologically active analogs or derivatives thereof (i.e. , those having a substantially equivalent biological activity). Known analogs and derivatives are disclosed, for examples in Islam et al., J. Med. Chem. 34 2954-61 , 1991 ; Skibo et al., J. Med. Chem. 37:78-92, 1994; Behroozi et al., Biochemistry 35:1568- 74, 1996; Helissey et al., Anticancer Drug Res. 11 :527-51 , 1996; llnno et al., Chem. Pharm. Bull. 45:125-33, 1997; llnno et al., Bioorg. Med. Chem., 5:903-19, 1997; llnno et al., Bioorg. Med. Chem., 5: 883-901 , 1997; and Xu et al., Biochemistry 37:1890-7, 1998). Other examples include, but are not limited to, endiyne quinone imines (U.S. Pat. No. 5,622,958); 2,2r-bis (2-aminoethyl)-4-4'-bithiazole [Lee et al., Biochem. Mol. Biol. Int. 40:151-7, 1996]; epilliticine-salen. copper conjugates [Routier et al., Bioconjug. Chem., 8: 789-92, 1997],

[0161] Some of the aforementioned chemotherapy agents can be grouped together under a common category as antimetabolites. “Antimetabolite” as used herein, refers to the compounds which inhibit the use of a metabolite that is part of normal metabolism. Antimetabolites are often similar in structure to the metabolite that they interfere with, such as the antifolates that interfere with the use of folic acid. Non-limiting examples of antimetabolites include the following compounds: bleomycin, busulfan, capecitabine, carmustine, carboplatin, chlorodeoxyadenosine, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate mitomycin, mitoxantrone, oxaliplatin, paclitaxel, procarbazine, SN-38, thioguanine, thiotepa, teniposide vinblastine, vincristine, and vinorelbine.

[0162] As used herein, the term “cytotoxic polypeptide” refers to an agent that is capable of inhibiting cell function. The agent may inhibit proliferation or may be toxic to cells. Any polypeptides that when interacting with a cell may interfere with or detrimentally alter cellular metabolism or in any manner inhibit cell growth or proliferation are included within the ambit of this term, including, but not limited to, agents whose toxic effects are mediated at the cell surface and also those whose toxic effects are mediated when transported into the cell. Useful cytotoxic polypeptides include proteinaceous toxins such as bacterial toxins.

[0163] Examples of proteinaceous cell toxins useful for incorporation into the fusion protein according to the invention include, but are not limited to, type one and type two ribosome inactivating proteins (RIP). Useful type one plant RIPs include, but are not limited to, dianthin 30, dianthin 32, lychnin, saporins 1-9, pokeweed activated protein (PAP), PAP II, PAP-R, PAP-S, PAP-C, mapalmin, dodecandrin, bryodin-L, bryodin, Colicin 1 and 2, luffin-A, luffin-B, luffin-S, 19K-protein synthesis inhibitory protein (PSI), 15K-PSI, 9K-PSI, alpha-kirilowin, beta-kirilowin, gelonin, momordin, momordin-ll, momordin-lc, MAP-30, alpha-momorcharin, beta-momorcharin, trichosanthin, TAP-29, trichokirin; barley RIP; flax RIP, tritin, corn RIP, Asparin 1 and 2 [Stirpe et al., 1992. Bio / Technology 10:405-12], Useful type two RIPs include, but are not limited to, volkensin, ricin, nigrin-b, CIP-29, abrin, modeccin, ebulitin-[alpha], ebulitin-[beta], ebultin-[gamma], vircumin, porrectin, as well as the biologically active enzymatic subunits thereof [Stirpe et al., 1992. Bio / Technology 10:405-12; Pastan et al., 1992. Annu. Rev. Biochem. 61 :331-54; Brinkmann and Pastan, 1994. Biochim. et Biophys. Acta 1198:27-45; and Sandvig and Van Deurs, 1996. Physiol. Rev. 76:949-66],

[0164] Examples of bacterial toxins useful as cell toxins include, but are not limited to, shiga toxin and shiga-like toxins (i.e. , toxins that have the same activity or structure), as well as the catalytic subunits and biologically functional fragments thereof. These bacterial toxins are also type two RIPs [Sandvig and Van Deurs, 1996. Physiol. Rev. 76:949-66; Armstrong, 1995. J. Infect. Dis., 171 :1042-5; Kim et al., 1997. Microbiol. Immunol. 41 :805-8; and Skinner et al., 1998. Microb. Pathog. 24:117-22], Additional examples of useful bacterial toxins include, but are not limited to, Pseudomonas exotoxin and Diphtheria toxin [Pastan et al., 1992. Annu. Rev. Biochem. 61 :331-54; and Brinkmann and Pastan, 1994. Biochim. et Biophys. Acta 1198:27-45], Truncated forms and mutants of the toxin enzymatic subunits also can be used as a cell toxin moiety (Pastan et al., Annu. Rev. Biochem. 61 :331-54; Brinkmann and Pastan, Biochim. et Biophys. Acta 1198:27-45, 1994; Mesri et al., J. Biol. Chem. 268:4852-62, 1993; Skinner et al., Microb. Pathog. 24:117-22, 1998; and U.S. Pat. No. 5,082,927). Other targeted agents include, but are not limited to the more than 34 described Colicin family of RNase toxins which include colicins A, B, D, E1-9, cloacin DF13 and the fungal RNase, [alpha]-sarcin [Ogawa et al. 1999. Science 283: 2097-100; Smarda et al., 1998. Folia Microbiol (Praha) 43:563- 82; Wool et al., 1992. Trends Biochem. Sci., 17: 266-69],

[0165] The term "anti-angiogenic polypeptide", as used herein, denotes a polypeptide capable of inhibiting angiogenesis. Suitable antiangiogenic polypeptides include, without limitation, angiostatin, endostatin, anti-angiogenic anti-thrombin III, sFRP-4 as described in W02007115376, and an anti-VEGF antibody such as anibizumab, bevacizumab (avastin), Fab IMC 1121 and F200 Fab. Proliferation of tumor cells relies heavily on extensive tumor vascularization, which accompanies cancer progression. Thus, inhibition of new blood vessel formation with anti-angiogenic agents and targeted destruction of existing blood vessels have been introduced as effective and relatively non-toxic approaches to tumor treatment.

[0166] As used herein, a "tumor suppressor" is a gene or gene product that has a normal biological role of restraining unregulated growth of a cell. The functional counterpart to a tumor suppressor is an oncogene — genes that promote normal cell growth may be known as "proto-oncogenes” A mutation that activates such a gene or gene product further converts it to an "oncogene", which continues the cell growth activity, but in a dysregulated manner Examples of tumor suppressor genes and gene products are well known in the literature and may include PTC, BRCA1 , BRCA2, p16, APC, RB, WTI, EXTI, p53, NFI, TSC2, NF2, VHL.ST7, ST14, PTEN, APC, CD95 or SPARC.

[0167] The term “pro-apoptotic polypeptides”, as used herein, refers to a protein which is capable of inducing cell death in a cell or cell population. The overexpression of these proteins involved in apoptosis displaces the careful balance between anti-apoptotic and pro-apoptotic factors towards an apoptotic outcome. Suitable pro-apoptotic polypeptides include, without limitation, pro-apoptotic members of the BCL-2 family of proteins such as BAX, BAK, BOK / MTD, BID, BAD, BIK / NBK, BLK, HRK, BIM / BOD, BNIP3, NIX, NOXA, PUMA, BMF, EGL-I, and viral homologs, caspases such as caspase-8, the adenovirus E4orf4 gene, p53 pathway genes, pro-apoptotic ligands such as TNF, FasL, TRAIL and / or their receptors, such as TNFR, Fas, TRAIL-R1 and TRAIL-R2. The term “metastasis suppressor” as used herein, refers to a protein that acts to slow or prevent metastases (secondary tumors) from spreading in the body of an organism with cancer. Suitable metastasis suppressor includes, without limitation, proteins such as BRMS I, CRSP3, DRGI, KAI1 , KISS-I, NM23, a TIMP-family protein and uteroglobin.

[0168] As used herein, an immunostimulatory polypeptide agent is a polypeptide encoded by a polynucleotide which is capable of activating or stimulating the immune response (including enhancing a pre-existing immune response) in a subject to whom it is administered, whether alone or in combination with another agent. Suitable non-limiting examples of immunostimulatory peptides include flagellin, muramyl dipeptide), cytokines including interleukins (e.g., IL-2, IL-7, IL- 15 (or superagonist / mutant forms of these cytokines), IL-12, IFN-gamma, IFN-alpha, GM-CSF, FLT3-ligand, etc.), immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3, or single chain / antibody fragments of these molecules), and the like.

[0169] As used herein, the term “toxins” refers to non-proteinaceous / non-polypeptidic cytotoxic compounds obtained from different organisms, as well as chemically modified derivatives of those same compounds and compounds obtained through chemical synthesis. The compounds of this category with biological origin may be obtained from microorganisms (whether bacteria, archaea, protozoa or unicellular fungi) or pluricellular organisms (pluricellular fungi, plants, or animals, like mollusks). It is intended that the chemical composition and structure of these toxins is not limited in any way beyond their non-polypeptidic nature, therefore one or more amino acids may be part of their structure, whether as part of their basic composition or as result of chemical derivation, as long as all the amino acids participating in the structure are not bound together by peptide bonds.

[0170] Examples of toxins suitable for the invention are calicheamycin y1 , dolastatin 10, maytansinoid (DM1) and pyrrolobenzodiazepine dimer (PBD).

[0171] In some embodiments, the second active domain is a marker agent.

[0172] The term "marker agent" refers to a marker molecule, for example a polypeptide, that is used to detect, identify or monitor the expression and / or localization and / or purification of the conjugate and is readily detectable in biological samples. Examples of marker agents include radioactive isotopes, fluorophores, fluorescent proteins, biotin, enzymatic polypeptides (such as horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase and p-galactosidase), polyhistidine polypeptides, maltose binding protein, Glutathione S-transferases, Myc protein, S-tag protein, V5 tag.

[0173] The term "radioactive isotope" as described herein refers to a radioactive nuclide, and commonly-used isotopes include lodine-131 , lodine-125, phosphorus-32 and the like, which can be conjugated to a protein or polypeptide.

[0174] The term "fluorophore" (also called fluorochrome) refers to a molecule that absorbs energy of a specific wavelength and emits it, with lower energy, in a determined one of greater wavelength. Examples of fluorophores are: fluorescein isothiocyanate (ITFC), ethidium bromide, propidium iodide, phycoerythrin, cyanine, phycocyanin, allophycocyanin, 6-FAM, Cy5, Cy3, TAMRA, JOE, MAX, TET, Cy5.5, ROX, TYE 563, Ykima Yellow, HEX, TEX 615, TYE 665, TYE 705, Alexa Fluorine (350, 405, 488, 532, 546, 555, 568, 594, 647, 660, 680, 750), LI-COR IR ( 700, 800, 800CW), ATTO (488, 532, 550, 565, Rho101 , 590, 633, 647N), Rhodamines (Green-X, Red-X, 5-TAMRA), WellRED (D4, D3, D2), Texas Red, Texas Red-X, Lightcycler 640, Dt 750, GFP, Oregon Green, Pacific Blue, Pacific Orange, Pacific Green, Coumarin, Tetramethylrhodamine (TRITC), BODIPY FL, Super Bright (436, 600, 645, 702), DAPI, SYTOX Green, SYTO 9, TO-PRO-3, Qdot (525, 565, 605, 655, 705, 800).

[0175] “Fluorescent protein”, as used herein, relates to proteins whose atomic structure allows them to present fluorescence, which is a phenomenon well-known in the art. Non-limiting examples of commonly used fluorescent proteins suitable for the fusion protein of the invention, are the green fluorescent protein (GFP, first discovered in Aequorea victoria), the red fluorescent protein (RFP), the yellow fluorescent protein (YFP), the blue fluorescent protein (BFP), the cyan fluorescent protein, or any other variant, examples of which can be found in Kremers et al. [Kremers, G-J- et al. 2011. J. Cell Sci. 124:157- 160],

[0176] Additional non-limiting examples of fluorescent proteins suitable for the conjugates or fusion protein of the invention are the enhanced green fluorescent protein (eGFP), enhanced cyan fluorescent protein CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilised ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T- Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed- monomer, J- Red, dimer2, t-dimer2(12), mRFPI, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B- Phycoerythrin, R-Phycoerythrin and Allophycocyanin. In other embodiments, the marker is a fluorescent protein selected from the group consisting of the mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum [Shaner et al. (2005) Nat. Methods 2:905-909], and the like.

[0177] In a preferred embodiment, the second active domain is a binding / targeting agent.

[0178] The term “binding / targeting agent”, as used herein, relates to an agent or domain that binds to a molecule of interest, thus targeting the conjugate to this molecule or to any cell which shows surface expression of said molecule.

[0179] In a preferred embodiment the binding / targeting agent is a ligand that binds E3 ligase, thus forming a PROTAC (proteolysis targeting chimera) which results in the recruitment of the E3 ligase to CD33 resulting in its ubiquitination and subsequent degradation.

[0180] In a preferred embodiment the binding / targeting agent is another antibody binding to a molecule of interest, thus forming a bi- or multi- specific antibody conjugate. In some embodiments, the second antibody is an antibody that binds specifically to a cell of the immune system thereby reinforcing the recruitment of the cells of the immune system to those cells in which CD33 is expressed. In some embodiments, the second antibody is an anti-CD3 antibody (suitable for targeting the conjugate to T cells), an anti-CD16 antibody (suitable for targeting the conjugate to NK cells), an antibody specific for the y or 6 chains of the T cell receptor (suitable for targeting the conjugate to Vy9V<52 T cells.

[0181] In some embodiments, the second domain of the conjugates according to the invention is an immune checkpoint inhibitor such as an anti-PD 1 checkpoint inhibitor (including, but not limited to Nivolumab (BMS-936558 or MDX1106), pembrolizumab, CT-011 , MK- 3475), an anti-PDL1 checkpoint inhibitor (including, but not limited to atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C).

[0182] Polynucleotides, vectors and host cells

[0183] The sdAbs, biparatopic sdAb and the conjugate, whenever the second domain is a polypeptide fused to the first domain, according to the invention can all be encoded by a polynucleotide. Therefore, another aspect of the present invention relates to a polynucleotide encoding the sdAb or the biparatopic sdAb according to the invention or the conjugate, whenever the second domain is a polypeptide fused to the first domain, according to the invention. The term “nucleic acid”, "nucleotide sequence", or "polynucleotide" is used interchangeably in the present invention to refer to the polymeric form of the ribonucleoside phosphate ester (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxy ribonucleosides (deoxiadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; "DNA molecules") or any phosphoester analog thereof such as phosphorothioates and thioesters, in a single stranded or double stranded form. Thus, the term includes single stranded DNA or RNA molecules. It also includes double stranded molecules formed by DNA-DNA, DNA-RNA and RNA-RNA strands. The term "nucleic acid sequence" and, in particular, the DNA or RNA molecule, refers only to the primary or secondary structure of the molecule and does not limit any particular type of tertiary structure. Thus, this term encompasses double stranded DNA, as comprised in linear or circular DNA molecules, supercoiled DNA plasmids and chromosomes. In a preferred embodiment, the nucleic acid is a DNA molecule. In another particular embodiment, it is an RNA molecule.

[0184] In a preferred embodiment the polynucleotide further comprises a sequence encoding a signal sequence which is fused in frame to the N-terminus of the sdAb or of the biparatopic sdAb.

[0185] The term “signal sequence”, also known as “leader peptide” or “signal peptide”, is used herein according to its ordinary meaning in the art and refers to a peptide having a length of about 5-30 amino acids. A leader peptide is present at the N-terminus of newly synthesized proteins that form part of the secretory pathway. Proteins of the secretory pathway include, but are not limited to proteins that reside either inside certain organelles (the endoplasmic reticulum, Golgi or endosomes), are secreted from the cell, or are inserted into a cellular membrane. In some embodiments, the leader peptide forms part of the transmembrane domain of a protein.

[0186] The polynucleotide of the invention can be present in a vector which can be used to express the polypeptide of the invention in different host cells. A further aspect of the present invention relates to a vector comprising the polynucleotide according to the invention.

[0187] The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In a particular embodiment, the vector of the invention is a cloning or expression vector. Suitable vectors for the insertion of the polynucleotide sequence of the invention are vectors derived from prokaryotic vectors such as plIC 18, plIC 19, pET28, pET15, pGEX-6P-l, Bluescript and their derivatives, mp I 8, mp I9, pBR322, pMB9, Col El, PCRI, RP4, phage and "shuttle" vectors such as pSA3 and pAT28, yeast expression vectors such as yeast 2 micron plasmid, integration plasmid, yeast episomalplasmid (YEp) vectors, similar and centromeric plasmids, expression vectors in insect cells such as vectors the pAC series and the pVL series, plant expression vectors such as vectors series pl Bl, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and similar and expression vectors in eukaryotic cells rather than based on viral vectors (adenovirus, adenovirus associated viruses and retroviruses, and particularly, lentivirus, especially those of third generation, based on transfer plasmid pCCL and packaging vectors pRSV-Rev, pMD2G, and pMDLg / pRRE) and non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg pHCMV / Zeo, pCR3.1 , pEFI / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, PTRACE- HCMV, pUB6N5-His, pVAXI, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1 , pML2d and pTDTI. By way of illustration, the vector in which said nucleic acid sequence is introduced can be a plasmid which is or is not integrated in the genome of a host cell when it is introduced in said cell. Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the invention or the gene construct of the invention can be inserted include a Tet-On inducible vector for expression in eukaryote cells. The vector of the invention can be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 1989). In a particular embodiment of the vector of the invention, said vector is a vector useful for transforming animal cells.

[0188] The vector of the invention can be a viral or non-viral vector, and can be used to transform, transfect, or infect cells which can be transformed, transfected, or infected by said vector. In this sense, another aspect of the present invention relates to a host cell comprising the polynucleotide according to the invention and / or the vector according to the invention and / or expressing the polypeptide of the invention. Said cells can be prokaryotic or eukaryotic. The vector of the invention can be used to transform eukaryotic cells such as yeast cells, Saccharomyces cerevisiae, or mammalian cells for example epithelial kidney 293 cells or LI2OS cells, or HeLa cells or prokaryotic cells such as bacteria, Escherichia coli or Bacillus subtilis, for example.

[0189] CD33-specific chimeric antigen receptor

[0190] The sdAb according to the invention can be used to obtain chimeric antigen receptors, which are useful in the treatment of cancer. Hence, another aspect of the present invention relates to a polynucleotide encoding for a CD33-specific chimeric antigen receptor (CAR) comprising: (a) a sdAb according to the invention or the biparatopic sdAb according to the invention;

[0191] (b) a transmembrane domain; and

[0192] (c) at least one intracellular signaling domain and / or costimulatory domain.

[0193] As used herein, a "chimeric antigen receptor” or “CAR" also known as chimeric T cell receptors, a T-body, artificial T cell receptors and chimeric immune receptors (CIR), are engineered receptors, which graft an arbitrary specificity onto an immune effector cell. In a classical CAR, the specificity of a monoclonal antibody is grafted on to a T cell. CARs are therefore fusion proteins which comprise at least, an extracellular domain or antigen binding domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from a polypeptide from which the extracellular domain is derived, and at least one intracellular signaling and / or costimulatory domain. In the present context the of CARs, the domain capable of binding to an antigen comprises or corresponds to a sdAb or biparatopic sdAb according to the invention which bind to CD33, thereby making the CAR specific for binding to CD33.

[0194] The second element of the polypeptide encoding a CD33-specific CAR is a transmembrane domain that is attached to the domain comprising the sdAb or the biparatopic sdAb.

[0195] As used herein, “transmembrane domain” (TMD) refers to the area of CAR that crosses the cell membrane. The transmembrane domain of the CAR expressed by the immune cell of the invention is the transmembrane domain of a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is one that is associated with one of the other domains of the CAR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In a preferred embodiment, the transmembrane domain of the CAR expressed by the immune cell of the invention is capable of homodimerization with another CAR on the CAR T cell surface. In a different particular embodiment of the CAR expressed by the immune cell of the invention, the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR T.

[0196] The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target. Non limiting examples or transmembrane domains of particular use in this invention may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD3 zeta, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD 278), 4-1 BB (CD137), GITR, CD40, CTLA4, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1 , VLA1 , CD49a, ITGA4, IA4 CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDGA, CDGA, CD103, ITGAL, CDLa, LFA-1 , ITGAM, CDIIb, ITGAX, CDIc, ITGB1 , CD29, ITGB2, CD18, LFA-1 , LGA ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), SLAMF6 (NTB-A) , LylOS), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, Kp30, NKp46, including NKG2D, and / or a transmembrane domain selected from the transmembrane domain of NKG2C.

[0197] The transmembrane domain may be recombinant, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In one aspect a triplet of phenylalanine, tryptophan and valine can be found at each end of a recombinant transmembrane domain.

[0198] In a preferred embodiment of the polynucleotide encoding a CD33-specific CAR, the transmembrane domain is selected from the group consisting of the CD4 transmembrane domain, the CD8a transmembrane domain, the CD28 transmembrane domain, the 4- 1 BB transmembrane domain, the CTLA4 transmembrane domain, the CD27 transmembrane domain and the CD3 zeta transmembrane domain. In another preferred embodiment of the polynucleotide encoding a CD33-specific CAR, the transmembrane domain is the CD8a transmembrane domain, preferably comprising or consisting of a sequence according to SEQ ID NO: 40.

[0199] In some instances, the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen binding domain of the CAR, via a hinge, preferably a hinge from a human protein. In a preferred embodiment, the polynucleotide encoding a CD33-specific CAR further comprises a hinge domain between the sdAb and the transmembrane domain. In another preferred embodiment, the hinge can be a human Ig (immunoglobulin) hinge, such as an lgG4 hinge, or a CD8a hinge.

[0200] As used herein, “hinge domain”, “hinge region” or “spacer” refers to an amino acid region that allows for separation and flexibility of the antigen domain (sdAb or the biparatopic sdAb) and the transmembrane domain, in effective allowing separation from the T cell membrane. The length of the flexible hinges also allows for better binding to relatively inaccessible epitopes, e.g., longer hinge domains are allowed for optimal binding. One skilled in the art will be able to determine the appropriate hinge for the given CAR target. Examples of hinge domains are human immunoglobulin (Ig) hinges, such as the lgG4 hinge, CD28 hinge or the CD8a hinge. In a preferred embodiment the hinge domain is selected from domain is selected from the group consisting of: CD8a, CD28, and lgG4 hinge domain. In a preferred embodiment the hinge domain is the CD8a hinge domain, is the cD28 hinge domain or is the lgG4 hinge domain. In another preferred embodiment the hinge domain comprises or consists of a sequence according to SEQ ID NO: 39.

[0201] The term “intracellular signaling domain” of the CD33-specific CAR as used herein, refers to the intracellular portion of a molecule and more specifically to any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell. The intracellular signaling domain generates a signal that stimulates the immune effector function of CAR-containing cells, for example, CAR-T cells. The effector function of a T cell, for example, may be cytolytic function or helper activity including the secretion of cytokines. Thus, the intracellular signaling domain may be a portion of a protein which transduces the effector function signal and directs the cell (e.g. T cell) to perform a specialized function.

[0202] Generally, the whole intracellular signaling domain can be used; however, it is appreciated that it is not necessary to use the entire domain, provided that whatever part of the signaling domain that is used is still capable of transducing the effector function signal. It will also be appreciated that variants of such intracellular signaling domains with substantially the same or greater functional capability may also be used. By this we include the meaning that the variants should have substantially the same or greater transduction of the effector functional signal. Typically, substantially the same or greater signal transduction includes at least 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120%, or more of the signal transduction of the unmodified intracellular signaling domain, wherein signal transduction of the unmodified intracellular signaling domain corresponds to 100%. Methods for assessing transduction of effector function signal are well known to those skilled in the art and include, for example, assessing the amounts and / or activity of molecules (e.g. proteins such as cytokines) that are indicative of the transduced signal. Thus, when the signal is the cytolytic function of a T-cell, the methods may involve measurement of one or more cytokines secreted by the T-cell, which cytokines are known to have a cytolytic activity (e.g. IFN gamma). Another means of assessing the cytolytic function is by CFSE staining and counting positive cells by Flow cytometry or by a chromium release assay as is well known in the art.

[0203] Examples of intracellular signaling domains for use in the CAR encoded by the polynucleotide according to the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0204] It is known that signals generated through the TCR alone are generally insufficient for full activation of a T cell and that a secondary and / or costimulatory signal may also be required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen- independent manner to provide a secondary or costimulatory signal (secondary intracellular signaling domain, such as a costimulatory domain). Costimulatory domains promote activation of effector functions and may also promote persistence of the effector function and / or survival of the cell.

[0205] In a preferred embodiment the at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or any combination thereof.

[0206] A primary intracellular signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs (e.g. 2, 3, 4, 5 or more ITAMs). Thus, the intracellular signaling domain may comprise one or more ITAMs. It will be appreciated that one or more ITAMs of the intracellular signaling domain may be modified, for example by mutation. The modification may be used to increase or decrease the signaling function of the ITAM as compared to the native ITAM domain.

[0207] Examples of ITAM containing primary intracellular signaling domains that are of particular use in the invention include those of CD3 zeta, Fc receptor gamma, Fc receptor beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment the at least one intracellular signaling domain of the CAR encoded by the polynucleotide according to the invention is selected from a group consisting of CD3 zeta, Fc receptor gamma, Fc receptor beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0208] As mentioned above, the intracellular signaling domain may comprise a primary intracellular signaling domain by itself, or it may comprise a primary intracellular signaling domain in combination with one or more secondary intracellular signaling domains, such as one or more costimulatory signaling domains. Thus, as an example, the intracellular signaling domain of the CAR encoded by the polynucleotide according to the invention may comprise the CD3 zeta signaling domain by itself or in combination with one or more other intracellular signaling domains such as one or more costimulatory signaling domains.

[0209] The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.

[0210] The term “co-stimulating molecule” refers to a recognizable T-cell binding partner that specifically binds to a co-stimulating ligand, thereby mediating the co-stimulatory response exerted by the T-cell, such as, but not limited to, proliferation. Co-stimulating molecules are cell surface molecules other than antigen-specific receptors or their ligands, which are necessary for an effective immune response. A costimulatory molecule may be a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of immune cells (e.g., lymphocytes) to an antigen. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins) and NK cell activation receptors. Examples of such molecules include, but are not limited to 0X40, ICOS, DAP10, CD27, CD28, CDS, CD30, CD137 (4-1 BB), CD40, ICOS, lymphocyte function- associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, GITR, NKG2C, SLAMF7, NKp80, BAFFR, HVEM, BTLA, ICAM-1 , LFA-1 (CD11a / CD18), B7- H3, and a ligand that specifically binds with CD83, and the like. For example, CD27 co- stimulation has been demonstrated to enhance expansion, effector function, and survival of human CAR T cells in vitro and augments human T cell persistence and anti-tumor activity in vivo (Song et al. Blood. 2012; 1 19(3):696-706).

[0211] In another preferred embodiment, the at least one intracellular signaling domain comprises the intracellular domain of the costimulatory molecules selected from 0X40, CD70, CD27, CD28, CD5, ICAM-1 , LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP 12, and 4-1 BB (CD137), or any combination thereof. In another preferred embodiment, the at least one intracellular signaling domain comprises the intracellular domain of 4- 1 BB. In another preferred embodiment, the at least one intracellular signaling domain comprises or consists of the sequence according to SEQ ID NO: 41.

[0212] In another preferred embodiment, the at least one intracellular domain further comprises a CD3 zeta intracellular domain.

[0213] The term “CD3” refers to the human CD3 protein complex, which is composed of six distinct chains: a CD3y chain (SwissProt P09693), a CD35 chain (SwissProt P04234), two CD3E chains (SwissProt P07766), and one CD3 chain homodimer (SwissProt P20963) (E y: e b:< ), and which is associated with the T cell receptor a and chain. The term includes any CD3 variants, isoforms and species homologs which are naturally expressed by cells, including T cells, or are expressed on cells transfected with genes or cDNA encoding the aforementioned chains.

[0214] The term “zeta” or alternatively “zeta chain”, “CD3 “CD3-zeta” or “TCR-zeta” is defined as the protein represented by GenBank entry No. BAG36664.1 , or equivalent residues from a non-human species, such as a mouse, rodent, monkey, primate, etc., and a “zeta stimulating domain” or alternatively a “CD3 zeta stimulating domain” or “TOR zeta stimulating domain” is defined as amino acid residues of the cytoplasmic domain of the zeta chain that are sufficient for functional transmission of the primary signal required to activate T cells etc. In one aspect, the zeta cytoplasmic domain comprises residues 52 through 164 inclusive of a GenBank entry protein of BAG36664.1 , or equivalent residues from a non-human species, for example, a mouse, rodent, monkey, primate, and the like, which are their functional orthologists.

[0215] In a preferred embodiment the CD3 zeta intracellular domain comprises or consists of the sequence according to SEQ ID NO: 42. The intracellular signaling sequences within the intracellular portion of the CAR encoded by the polynucleotide may be linked to each other in a random or specified order. In a preferred embodiment the at least one intracellular signaling domain is located N- terminally with respect to the CD3 zeta intracellular domain.

[0216] Optionally, a short oligo- or polypeptide linker as previously defined in relation to the biparatopic sdAb, may form the linkage between intracellular signaling sequences. In one embodiment, a glycine- serine doublet can be used as a suitable linker. In another embodiment, a single amino acid, such as an alanine or a glycine, can be used as a suitable linker.

[0217] In one embodiment, the intracellular signaling domain is designed to comprise two or more, for example 3, 4, 5, or more, costimulatory signaling domains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains, are separated by a linker molecule, such as one described herein. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0218] In a preferred embodiment the polynucleotide encodes a CD33-specific CAR wherein the hinge domain is the CD8a hinge domain, the transmembrane domain is the CD8a transmembrane domain, the intracellular signaling domain comprises the 4-1 BB intracellular region and the CD3 zeta cytoplasmatic domain.

[0219] In a further preferred embodiment, the CAR encoded by the polynucleotide further comprises a signal peptide located N-terminal to the sdAb or to the fusion protein.

[0220] The term “signal peptide” has been previously defined. In a preferred embodiment the signal peptide is the CD8 signal peptide and / or the GM-CSF receptor A signal peptide. In another preferred embodiment, the signal peptide comprises or consists of the sequence according to SEQ ID NO: 38 and / or SEQ ID NO: 44.

[0221] In a preferred embodiment the CD33-specific CAR encoded by the polynucleotide according to the invention comprises or consists of a sequence selected from SEQ ID NO: 46-51 and SEQ ID NO: 65.

[0222] In addition to the domains already mentioned, the CD33-specific CAR can comprise additional domains which facilitate its detection / identification / purification. In a preferred embodiment of the invention, the chimeric antigen receptor, encoded by the polynucleotide, further comprises a detection marker at the C-terminus, wherein the detection marker is connected to the chimeric receptor by a self-cleavable protein sequence.

[0223] In addition to the marker polypeptides previously defined and which are also of usability herein, the CAR encoded by the polynucleotide according to the invention, may further contain “markers”, “labels” or “tags” which may improve or facilitate certain properties of the protein, i.e. protein stability or resistance to degradation, or provide technical advantages during production, i.e., facilitate expression and / or purification. Examples of such tags are biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, fluorescent protein tag and the like. Said tags can be fused or conjugated to the amino-terminal or the carboxyl-terminal a polypeptide. His tags or “polyhistidines” sequence of histidine amino acids bound by peptide bonds which are fused or conjugated to the amino-terminal end of the protein of the invention, and / or to the carboxyl terminal end of the protein of the invention. In a preferred embodiment the polyhistidine region contains at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 histidine residues.

[0224] Another example of a detection marker is the truncated human EGFR polypeptide (huEGFRt) which is devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity but retains the native amino acid sequence, type I transmembrane cell surface localization, and a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab. huEGFRt can serve a highly efficient selection epitope for CAR-T cells as disclosed in Wang, X. et al. (2011 , Blood, 118(5): 1255-1263).

[0225] As used herein, the term "self-cleaving protein region" means a peptide sequence having a cleavage activity that occurs between two amino acid residues in the peptide sequence itself. Examples of the self-cleaving peptide include a 2A peptide and a 2A-like peptide. For example, in 2A or 2A-like peptides, cleavage occurs between glycine and proline residues on these peptides. This is caused by the "ribosome skipping mechanism", which prevents the formation of normal peptide bonds between glycine residues and proline residues during translation, and does not affect downstream translation. The ribosome skip mechanism is known in the art and is used for the expression of multiple proteins encoded by a single molecule of mRNA. The self-cleaving peptide used in the present invention can be obtained from a viral 2A peptide or a 2A-like peptide having an equivalent function. Examples of 2A self-cleaving peptides include 2A peptides (F2A) derived from foot-and-mouth disease virus (FMDV) (VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 56)), 2A peptides (E2A) derived from horse rhinitis A virus (ERAV) (QCTNYALLKLAGDVESNPGP (SEQ ID NO: 57)), 2A peptide (P2A) derived from Porcine teschovirus (PTV-1) (ATNFSLLKQAGDVEENPGP (SEQ ID NO: 58)), and 2A peptides (T2A) derived from Thosea signa virus (TaV) (EGRGSLLTCGDVEENPGP (SEQ ID NO: 59)). Mutations may be appropriately introduced into the self-cleaving peptide domain as long as its activity is not significantly impaired.

[0226] In a preferred embodiment the detection marker is huEGFRt and the self-cleavable protein sequence is the T2A peptide. In a more preferred embodiment, the detection marker is huEGFRt according to SEQ ID NO: 45 and the self-cleavable protein sequence is the T2A peptide according to sequence SEQ ID NO: 43.

[0227] The polynucleotide which encodes for the CD33-specific CAR can be present in a vector. Therefore, another aspect of the present invention relates to a vector which comprises the polynucleotide encoding a CD33-specific chimeric receptor antigen according to the invention.

[0228] Another aspect of the present invention relates to a polypeptide encoded by the polynucleotide encoding a CD33-specific chimeric receptor antigen according to the invention or by the vector comprising said polynucleotide encoding a CD33-specific chimeric receptor antigen according to the invention.

[0229] A further aspect relates to a cell comprising the polynucleotide encoding a CD33-specific chimeric receptor antigen according to the invention or the vector comprising said polynucleotide according to the invention. In a preferred embodiment the cell is characterized in that it expresses the chimeric antigen receptor encoded by the polynucleotide according to the invention. In another preferred embodiment, said cell is an autologous cell or an allogenic cell. In a further preferred embodiment said cell is further modified to silence the Human Leucocyte Antigen (HLA) Class I molecules and / or the T Cell Receptor (TCR). In a further preferred embodiment said cell is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell.

[0230] As used herein, the term “autologous” refers to cells which are obtained from the same organism to which they are going to be delivered to. For example, in the context of CAR- T cell therapy, autologous T cells are obtained from the subject being treated with the modified CAR-T cells. The term “allogenic” refers to cells which are obtained from a different organism to the one which they are going to be delivered to. As used herein, the term “Human Leucocyte Antigen”, or its acronym “HLA”, refers to a gene complex encoding the major histocompatibility complex (MHC) proteins in humans and refers to the histo-compatibility antigen systems in humans. The term “Human Leucocyte Antigen (HLA) Class I molecules” refers to a gene involved in regulating the expression of the HLA-I complex. The HLA-I complex is composed of an a chain encoded by an HLA-I gene and a p chain encoded by a B2M gene through non-covalent bonding. The HLA-I complex is expressed in most types of cells in the body, and can present endogenous antigen peptides (mostly 8-12 amino acids in length) to CD8+T cells and induce their killing function. HLA-I genes mainly include HLA-A, HLA-B, and HLA-C. In the context of the present invention, the expression “silence HLA Class I molecules” includes the partial or total reduction of the expression, partial or total deletion, or alteration of any one of the endogenous HLA-I related genes such that the HLA-I complex cannot perform its function. In other words, the expression level of any endogenous HLA-l-related gene in the cell is altered (e.g., inhibited, knocked down, knocked out, or silenced) by any gene editing or other non-gene editing means known in the field. In another preferred embodiment, the cell is further modified by having the B2M gene according to accession number 567 of the NCBI Gene database, version of August 19, 2025, silenced.

[0231] In the context of the present invention, the expression “silence TCR” includes the partial or total reduction of the expression, partial or total deletion, or alteration of any one of the endogenous TCR related genes (TRAC and TRBC genes) such that the TCR cannot perform its function. In another preferred embodiment, the cell is further modified by having the TRAC gene according to accession number 28755 of the NCBI Gene database, version of August 19, 2025, silenced.

[0232] In another preferred embodiment, the cell is further modified by having the TRAC gene according to accession number 28755 of the NCBI Gene database, version of August 19, 2025, and the B2M gene according to accession number 567 of the NCBI Gene database, version of August 19, 2025, both silenced.

[0233] As used herein, “immune cell” refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the cell is a T cell; a NK cell; a NKT cell; lymphocytes, such as B cells and T cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In a preferred embodiment the immune cell is selected from a group consisting of a T cell, a B cell, a NK cell, a neutrophil, an eosinophil, a basophil, a monocyte, a macrophage, a dendritic cell and a mast cell, or a precursor of any of these cells.

[0234] In a preferred embodiment the immune cell is a T cell and a precursor thereof.

[0235] As used herein, the term “T cell” refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes such as B lymphocytes by the presence of T cell receptors on the cell surface. T cells can also be isolated or obtained from commercially available sources. T cells are of any type expressing CD3, including primary T cells, helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, regulatory T cells (Tregs) and gamma-delta T cells. “Cytotoxic cells” include CD8+ T cells, natural-killer (NK) cells, and neutrophils capable of mediating a cytotoxic response. The terms "T cell" and "T lymphocyte" are interchangeable and are used interchangeably herein.

[0236] In a preferred embodiment the T cell is a primary T cell. The term “primary T cell” as used herein refers to any T-cell which can be obtained from an organism in possession of T-cells.

[0237] In a preferred embodiment the T cell is a natural killer cell.

[0238] Natural killer cells or “NK cells” are well known in the art. In one embodiment, natural killer cells include cell lines, such as NK- 92 cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1 , YTS cells, and NKL cells. NK cells can be detected by specific surface markers, such as CD16, CD56, and CD8 in humans. NK cells do not express T-cell antigen receptors, the pan T marker CD3, or surface immunoglobulin B cell receptors.

[0239] Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. Thus, NKT cells are a subset of T cells that coexpress an op T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1. Many of these cells recognize the non- polymorphic CD1d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. They constitute only approximately 0.1 % of all peripheral blood T cells. Natural killer T cells should not be confused with natural killer cells.

[0240] In a preferred embodiment the T cell is a Jurkat T cell. The term “Jurkat T cell” as used herein refers to an immortalized line of human T lymphocyte cells that are used to study acute T cell leukemia, T cell signaling, and the expression of various chemokine receptors susceptible to viral entry, particularly HIV.

[0241] As used herein, the term "somatic cell" refers to any cell other than germ cells, such as an egg, a sperm, or the like, which does not directly transfer its DNA to the next generation. Typically, somatic cells have limited or no pluripotency. Somatic cells used herein may be naturally-occurring or genetically modified.

[0242] As used herein, the term “progenitor cell” means a cell which is able to differentiate to form one or more types of cells but has limited self-renewal in vitro.

[0243] In order to obtain the cells according to the invention comprising the polynucleotide encoding a CD33-specific chimeric receptor antigen, said cells must be transduced with the polynucleotide according to the invention. As such, a further aspect of the present invention relates to an ex vivo method for obtaining a cell according to the invention comprising transducing the cell or precursor thereof with a polynucleotide encoding a CD33-specific chimeric receptor antigen according to the invention or with a vector according to the invention comprising said polynucleotide.

[0244] The term "transducing" refers to a process by which an exogenous polynucleotide is introduced into a host cell. A "transduced" cell is one which has been transduced with an exogenous polynucleotide. The cell includes the primary subject cell and its progeny.

[0245] Pharmaceutical compositions

[0246] The skilled person in the art will recognized that the sdAbs, biparatopic sdAbs, conjugates and CAR cells according to the invention can be accompanied by a pharmaceutical acceptable carrier. Therefore another aspect of the present invention relates to a pharmaceutical composition comprising the sdAb according to the invention, the biparatopic sdAb according to the invention, the conjugate according to the invention, the polynucleotide according to the invention, the vector according to the invention, the polynucleotide encoding a CD33-specific chimeric receptor antigen according to the invention, the vector according to the invention comprising the polynucleotide encoding a CD33-specific chimeric receptor antigen and / or the cell according to the invention comprising the polynucleotide encoding a CD33-specific chimeric receptor antigen and / or the vector comprising said polynucleotide.

[0247] As used herein, the term "pharmaceutically acceptable carrier" means a non- toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Remington's Pharmaceutical Sciences. Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995 discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[0248] The pharmaceutical compositions of this invention can be administered to a patient by any means known in the art including oral and parenteral routes. According to such embodiments, inventive compositions may be administered by injection (e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection), rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as by sprays).

[0249] Medicinal and treatment uses

[0250] Another aspect of the present invention relates to the sdAb according to the invention, the biparatopic sdAb according to the invention, the conjugate according to the invention, the polynucleotide according to the invention, the vector according to the invention comprising said polynucleotide, and / or the cell according to the invention comprising said vector or polynucleotide for use in medicine, wherein the polynucleotide encodes for the sdAb, biparatopic sdAb, conjugate, whenever the conjugate is a fusion protein, or the CD33-specific CAR according to the invention.

[0251] Another aspect relates to the sdAb according to the invention, the biparatopic sdAb according to the invention, the conjugate according to the invention, the polynucleotide according to the invention, the vector according to the invention comprising said polynucleotide, and / or the cell according to the invention comprising said vector or polynucleotide for use in the treatment of cancer, wherein the polynucleotide encodes for the sdAb, biparatopic sdAb, conjugate, whenever the conjugate is a fusion protein, or the CD33-specific CAR according to the invention.

[0252] Another aspect relates to a method for the treatment of cancer in a patient in need thereof which comprises the administration to the subject the sdAb according to the invention, the biparatopic sdAb according to the invention, the conjugate according to the invention, the polynucleotide according to the invention, the vector according to the invention comprising said polynucleotide, and / or the cell according to the invention comprising said vector or polynucleotide for use in the treatment of cancer, wherein the polynucleotide encodes for the sdAb, biparatopic sdAb, conjugate, whenever the conjugate is a fusion protein, or the CD33-specific CAR according to the invention.

[0253] As used herein, the terms "treat", "treatment" and "treating" refer to the reduction or amelioration of the progression, severity and / or duration of cancer, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of cancer. The terms "treat", "treatment" and "treating" also refer to the amelioration of at least one measurable physical parameter of cancer, such as growth of a tumor, not necessarily discernible by the patient. Furthermore, "treat", "treatment" and "treating" refer also to the inhibition of the progression of cancer, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. "Treat", "treatment" and "treating" may refer, too, to the reduction or stabilization of tumor size or cancerous cell count.

[0254] The term “cancer” refers to a group of diseases involving abnormal, uncontrolled cell growth and proliferation (neoplasia) with the potential to invade or spread (metastasize) to other tissues, organs or, in general, distant parts of the organism; metastasis is one of the hallmarks of the malignancy of cancer and cancerous tumors. The abnormal growth and / or proliferation of cancerous cells is the result of a combination of genetic and environmental factors that alter their normal physiology. The growth and / or proliferation abnormalities of cancerous cells result in physiological disorders and, in many cases, death of the individual, due to the dysfunctionality or loss of functionality of the cell types, tissues and organs affected.

[0255] The term “cancer” includes, but is not restricted to, cancer of the breast, heart, small intestine, colon, spleen, kidney, bladder, head, neck, ovaries, prostate gland, brain, pancreas, skin, bone, bone marrow, blood, thymus, womb, testicles, hepatobiliary system and liver; in addition to tumors such as, but not limited to, adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, hepatobiliary cancer, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma and teratoma. Furthermore, this term includes acrolentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, Bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Ewing sarcoma, focal nodular hyperplasia, germ cell tumors, glioblastoma, glucagonoma, hemangioblastoma, hemagioendothelioma, hemagioma, hepatic adenoma, hepatic adenomastosis, hepatocellular carcinoma, hepatobilliary cancer, insulinoma, intraepithelial neoplasia, squamous cell intraepithelial neoplasia, invasive squamous-cell carcinoma, large cell carcinoma, leiomyosarcoma, melanoma, malignant melonoma, malignant mesothelial tumor, medulobastoma, medulloepithelioma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, papillary serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, microcytic carcinoma, soft tissue carcinoma, somatostatin secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, Wilm tumor, intracerebral cancer, head and neck cancer, rectal cancer, astrocytoma, glioblastoma, microcytic cancer and non-microcytic cancer, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer and breast cancer.

[0256] In a preferred embodiment of the invention, the cancer is an hematological malignancy

[0257] The term "hematological malignancy" includes lymphoma, leukemia, acute myeloid leukemia, myeloma or lymphoma, as well as spleen cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphoma and T-cell lymphoma. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphoma includes, for example, cutaneous T-cell lymphoma. Hematological malignancies also include leukemias, such as secondary leukemia or acute lymphocytic leukemia. Hematological malignancies also include myeloma (eg, multiple myeloma) and other hematological and I or B-cell or T- cell-related cancers. In a preferred embodiment according to the invention the hematological malignancy is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute B cell lymphoblastic leukemia, minimal residual disease (MRD)- positive ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), lymphoplasmacytic lymphoma (LPL), B cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt’s lymphoma (BL), primary mediastinal large B-cell lymphoma (PMBL), marginal zone B cell lymphoma, Hodgkin’s lymphoma (HL), nonHodgkin’s lymphoma (NHL), NK- and T-cell neoplasms, histiocytic neoplasms, mantle cell lymphoma (MCL), hairy cell leukemia (HCL), plasma cell myeloma (PCM), plasma cell leukemia (PCL), and multiple myeloma (MM). The sdAbs, biparatopic sdAbs, conjugates, and CAR cells are all characterized by having binding specificity for human CD33. Therefore, in a preferred embodiment of the present invention, the hematological malignancy is characterized by the presence of malignant cells which exhibit CD33 expression in the surface.

[0258] The expression “CD33 expression in the surface” as used herein refers to cells which express CD33 protein and which display the extracellular N-terminal region of CD33 protein, comprising two immunoglobulin domains, on the surface of the plasma membrane towards the exterior medium. The N-terminal region is attached to the membrane by the transmembrane domain of CD33 and further comprises a C-terminal domain which is intracellular. CD33 is naturally expressed in cells from the myeloid lineage.

[0259] The identification and / or quantification of cells which exhibit CD33 expression in their surface can be carried out by known techniques in the art such as, without limitation, as specific antibody-binding capacity (SABC) units using multidimensional flow cytometry, flow cytometry and IHC staining,

[0260] In a preferred embodiment of the present invention, the malignant cells which exhibit CD33 expression in the surface are malignant blast cells. In another preferred embodiment of the present invention, the malignant cells exhibit at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more CD33 expression in the surface when compared to the expression exhibited by nonmalignant cells which express CD33 in their surface.

[0261] Analytical methods according to the invention

[0262] The skilled person in the art will recognize that the sdAbs according to the invention find utility in method to detect the presence or level of CD33 in a sample. Hence, another aspect of the present invention relates to a method to detect the presence or level of CD33 in a sample comprising:

[0263] (a) contacting the sample with the sdAb according to the invention or the biparatopic sdAb according to the invention or the conjugate according to the invention so as to obtain a mixture wherein said sdAb, biparatopic sdAb or conjugate is allowed to substantially bind with said CD33 when the latter is present in said sample; and (b) detecting the binding of the sdAb or of the biparatopic sdAb to the CD33 present in the sample thereby providing an indication of the presence or of the level of CD33 in the sample.

[0264] The term “sample” or “biological sample”, as used herein, refers to biological material isolated from a subject. The biological sample contains any biological material suitable for detecting the desired protein markers. The biological sample can comprise cell and / or non-cell material of the subject. The sample can be isolated from any suitable tissue or biological fluid such as, for example blood, saliva, cerebrospinal fluid, urine, stool, bone marrow, a nipple aspirate, a solid tumor biopsy, plasma, serum, cerebrospinal liquid (CSF), feces, a buccal or buccal-pharyngeal swab, a surgical specimen, a specimen obtained from a biopsy, and a tissue sample embedded in paraffin. Methods for isolating samples are well known to those skilled in the art. In a particular embodiment, the sample comprises cells expressing CD33.

[0265] The first step of the method to detect the presence or level of CD33 in a sample comprises the mixing the sdAbs, the biparatopic sdAb or the conjugates according to the invention, with a sample such that the sdAbs, biparatopic sdAbs or conjugates are capable of binding the CD33, if present in the sample. In order to perform such a method, the expert is aware that the incubation time and parameters, such as temperature and agitation amongst others, must be adapted to the type of sample and the sdAbs used. In a preferred embodiment step (a) of the method is carried out with a labelled sdAb, biparatopic sdAb or conjugate which comprises a marker agent in the second domain.

[0266] In a further preferred embodiment step (a) is carried out with a sdAb, a biparatopic sdAb or a conjugate bound to a solid support. The term “solid support” as used herein refers to a material having a rigid or semi-rigid surface. Such materials will preferably take the form of small beads, pellets, disks, chips, or wafers, although other forms may be used. Such surfaces include, simply by way of example, surfaces of art-known supports such as beads, plates, cuvettes, filters, titer plates, and the like, that have avidin, streptavidin and / or any art known derivative of these agents linked or coated to the surface(s) of those supports. The supports are generally made of conventional materials, e.g., plastic polymers, cellulose, glass, ceramic, stainless steel alloy, and the like.

[0267] Step (b) of the method to detect the presence or level of CD33 in a sample comprises the detection of the binding of the sdAbs in the sample due to their binding to the CD33 present in the sample. Such a step can be carried out by the detection of a label such as the ones previously defined. In one hand, wherein in step (a) the sdAb, biparatopic sdAb or conjugate is labelled with a directly readable label, or wherein the marker agent present in the conjugate is directly readable, examples of such labels or markers are, without limitation, radionuclide, a fluorescent dye, a luminescent substance, then step (b) is carried out directly by reading the output of said detectable label. On another hand, wherein in step (a) the sdAb, biparatopic sdAb, or conjugate is not labelled with a directly readable label, then a second detecting molecule is required in order to be able to detect the binding of the sdAb, biparatopic sdAb or conjugate to the CD33 present in the sample. As such, in a preferred embodiment step (b) is carried out by further incubating the sample with one or more molecule which can identify the binding of the sdAb, biparatopic sdAb, or conjugate to the CD33, wherein the molecule comprises a detectable label. In another preferred embodiment the one or more detectable label is selected from an enzyme, a radionuclide, a fluorescent dye, a luminescent substance or biotin. In another preferred embodiment the one or more molecule of step (b) recognizes the non-directly readable label present in the sdAb, biparatopic sdAb, or conjugate according to step (a).

[0268] In another preferred embodiment of the method of the invention, step (b) of the method to detect the presence or level of CD33 in a sample is carried out by a method selected from the group consisting of: mass spectrometry, western blot, flow cytometry, fluorescence microscopy, immunoprecipitation, Enzyme-linked immunosorbent assay (ELISA), Immunoelectrophoresis, and affinity purification.

[0269] The method to detect the presence or level of CD33 in a sample according to the invention can also be used to the diagnosis of a disease characterized in increase in cells expressing CD33 in a patient. As such, another aspect of the present invention relates to a method for the diagnosis of a disease characterized by an increased level of cells expressing CD33 in a patient, the method comprising detecting the level of cells expressing CD33 in a sample from the patient by a method to detect the presence or level of CD33 in a sample according to the invention, wherein the presence of an increased number of cells expressing CD33 in the sample from the patient with respect to a reference value is indicative of the subject suffering from a disease characterized by an increased level of cells expressing CD33.

[0270] The term "subject" or "patient", as used herein, refers to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans; for example, human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats or rodents. Preferably, the subject is a male or female human of any age or ethnicity.

[0271] The term “diagnosed”, as used herein, refers to the determination and / or identification of a disease in a subject, i.e. the opinion reached about the disease state of a subject, i.e. the diagnostic opinion. As such, it can also be regarded as an attempt to classify individuals depending on their disease condition. As it will be understood by those skilled in the art, the diagnosis of a disease, although preferred to be, need not be correct for 100% of the subjects to be diagnosed or evaluated. The term, however, requires that a statistically significant portion of subjects identified as such are suffering from said disease. Whether a subject is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. The p-values are, preferably, 0.05, 0.01 , 0.005 or lower.

[0272] The expression “reference value” refers to a laboratory value used as reference for the values / data obtained by means of samples obtained from patients. The reference value or reference level can be an absolute value, a relative value, a value that has an upper and / or lower limit, a series of values, an average value, a median, a mean value, or a value expressed by referring to a control or reference value. A reference value may be based on the value obtained from a group of patients considered to be representative, such as the values obtained in a population of subjects from a chronological age group coinciding with that of the patient object of the study or based on a set of inclusion or exclusion samples of the sample to be analyzed. Alternatively, a reference value may be based on an individual sample, such as, for example, a value obtained from a sample from the patient object of the study, but obtained at a previous point in time.

[0273] The “reference value” can further be considered as a normal level of the number of cells expressing CD33 in their surface in a subject or patient which is considered to be healthy.

[0274] In a preferred embodiment the method of diagnose wherein the disease characterized by an increase level of cells expressing CD33 is cancer, preferably an hematological malignancy, more preferably acute myeloid leukemia.

[0275] *** The invention will be described by way of the following examples which are to be considered as merely illustrative and not limitative of the scope of the invention.

[0276] EXAMPLES

[0277] MATERIALS AND METHODS

[0278] Camelid immunization

[0279] Four young llamas (Lama glama) were immunized with several targets of interest, using 4-5 antigens per animal. A male llama (N°124) was immunized with the CD33 recombinant ectodomain (SinoBiological #12238-H08H), and other antigens, four times at intervals of 20-30 days between doses. The first dose consisted of a subcutaneous injection of 150 pg of antigen with Freund’s complete adjuvant, while the boosters comprised subcutaneous administration of 200 pg of CD33 with incomplete Freund’s adjuvant, alternating flanks of immunization. Before each administration, a small blood sample (1-5 mL) was extracted for immune response evaluation. Indirect Enzyme-linked immunosorbent assay (ELISA) was performed to assess the anti-CD33 antibody titter in plasma at each timepoint. For this purpose, high-binding 96-well plates (Corning #3590) were coated with 0.3 pg / well of CD33 overnight at 4°C and blocked with 5% skimmed powdered milk before adding 1 serial dilutions of the plasma samples. After 1 hour of incubation at room temperature, plates were washed five times with PBS-tween 20 (0.05%) and then incubated with 100 pL / well of a 1 / 10000 dilution of the Goat anti-Llama IgG (H+L) Secondary Antibody, HRP (Invitrogen #A16060) for 1 hour. After eight washes, HRP was revealed with 100 pL of TMB substrate solution, and the reaction was stopped with 50 pL of sulfuric acid. Sigmoidal curves were graphed for EC50 determination using GraphPad Prism software. sdAb phage display library construction

[0280] After immunization, 100 mL of peripheral blood was extracted from the llama and transported in a 1 :4 proportion of 3.2% sodium citrate as an anticoagulant. Following a 1 :1 dilution with PBS, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (15 mL of Ficoll for each 25 mL of blood) and washed with PBS. After counting in a Neubauer's chamber, three mL of TRIzol reagent was added per million PBMCs, and the mixture was stored at -80°C. RNA was isolated via the TRIzol-chloroform method in Phasemaker™ tubes (Invitrogen #A33248), and RNA quality was evaluated by observing the 28S and 18S rRNA subunits on 1 % agarose gel electrophoresis. cDNA was generated from the extracted RNA using the RevertAid first-strand cDNA synthesis kit (Thermo Scientific #K1621), and sdAb DNA was obtained by PCR amplification using VH1 , VH3, and VH4 forward primers and JH reverse primers with the Kapa TAQ kit (Roche #KK1015). The sdAb DNA repertoire of each llama was digested with Sfil enzyme (NEB #R0123L) following the provider's instructions. Digested DNA was purified and ligated into digested and dephosphorylated pCOMB3XSS phagemid vector (Addgene #63890) using T4 DNA ligase (Thermo Scientific #EL0012) in a 1 :3 ratio of vector to insert. Electrocompetent E. coli TG1 (LGC #60502-2) and ER2738 (LCG #60522-2) were transformed with the ligation products and recovered in SOO medium. Transformants were selected with ampicillin before being infected with M13KO7 helper phage (NEB #N0315) for 2 hours, followed by selection with kanamycin. The culture was grown overnight at 37°C, and phages were obtained via PEG8000-NaCI double precipitation of the culture supernatant. The final phage libraries were titrated by infecting E. coli ER2378 and plate counting.

[0281] CD33 binder isolation: Panning and screening

[0282] To select CD33-specific binders, three rounds of panning were carried out in two 96-well high-binding plates coated with 1 pg of CD33 recombinant ectodomain overnight at 4°C. The coated wells were then blocked with 1% BSA in PBS, along with two additional uncoated wells, to perform a “pre-panning” incubation to clear out potential BSA binders. A total of 2 x 1011pfu of the phage libraries in ER2738 and TG1 in 100 pL of PBS with 1 % BSA were incubated on the uncoated BSA-blocked wells ("pre-panning" step) to eliminate potential binders to BSA or to the plate surface, for 30 minutes at RT. The cleared phage supernatant was then added to the CD33-coated wells and incubated for 1 hour at room temperature. Supernatant containing non-binders was eliminated, and the plate was washed 25 times with PBS containing 0.05% Tween 20, in cycles of 5 washes with 5 minutes of incubation in the last wash of each cycle. Binders were eluted using 10 mg / mL bovine pancreatic trypsin (Sigma #T4799256) and incubated for 30 minutes at 37°C. Protease inhibitor cocktail (Cell Signalling #5871 S) were added to the output phages, and ER2738 cells were infected for titration of the output phages and for phage amplification using helper phage M13KO7.

[0283] In subsequent rounds, the amplified output phages were used as input, maintaining the input at 2 x 1011pfu so that the output / input phage ratio could be used to indicate phage enrichment over the rounds. Additionally, phage ELISA was performed by offering 2 x 1011pfu of each output and the library and incubating with anti-M13 (HRP) antibody (SinoBiological #11973-MM05T-H). Screening of positive clones was conducted from two sources: the phagemid containing isolated clones (pCOMB3XSS) and the further subcloned clones containing the sdAbs in a pET-derived expression vector named pETMod. Single colonies from the titration plates of outputs 2 and 3 were grown and induced with 2 mM IPTG for sdAb expression, which typically leaks sdAb into the culture supernatant. Individual supernatants were added to ELISA plates coated with 0.25 pg / well of CD33 and blocked with PBS-BSA 1% (diluted 1 :1 on PBS-BSA 1%) and incubated for 1 hour at room temperature with shaking. After 5 washes with PBS-Tween 20 (0.05%), a 1 / 500 dilution of anti-HA HRP secondary antibody (Roche #11667475001) was added to each well. After 8 washes, HRP was revealed using Opti-EIA substrate reagents A and B at a 1 :1 ratio (BD #51-2606KC, #51- 2607KC). It is important to note that the resulting signal at this stage depends not only on sdAb affinities for CD33 but also on expression levels in bacteria, rendering this outcome a qualitative measurement.

[0284] CD33 binder subcloning in expression vector and sequencing

[0285] Once enrichment reached more than 80% of positive clones, the VHH repertoire of the last output was amplified in E. coli, and DNA extraction was performed using the Qiaprep plasmid kit (Qiagen #27106). The phagemid repertoire was then digested with Sfil for further cloning into the pETMod vector, which has been modified previously for sdAb expression and includes the periplasmic expression signal peptide OmpA. Ligation in a 1 :3 ratio of digested vector and insert was carried out using T4 DNA ligase. The cloned repertoire was used to transform E. coli BL21 DE3 (Novagen #69450-4), and single colonies selected with kanamycin (50 pg / mL on LB Agar plates) were isolated and evaluated for binding to CD33 as previously described. Plasmid DNA of each clone was then extracted and sequenced using the Sanger method with the ompseq forward primer (5’-AAGACAGCTATCGCGATTGCAG-3’ - SEQ ID NO: 61).

[0286] Expression and purification of anti-CD33 sdAbs

[0287] Following the sequencing of the positive clones, a multiple alignment of the sdAbs was performed using Jalview and Clustal Omega (EMBL-EBI), and a phylogenetic tree was constructed to identify sequences with the highest distance. SdAb candidates representing each cluster / family were selected for production and characterization, under the assumption that similar CDR sequences often interact with the same epitope in the antigen, while different ones are more likely to bind different epitopes, giving the sdAbs distinct properties. Candidate sdAbs were produced by culturing 500 mL of LB medium (Condalab #123100) inoculated with 1 % of an overnight seed culture of each isolated clone and supplemented with 50 pg / mL kanamycin. When the OD600 reached 0.6-1 , sdAb expression was induced with 1 mM IPTG for 4 hours at 37°C or overnight at 18°C, according to optimized conditions. Cultures were centrifuged at 3000 g for 30 minutes, and the bacterial pellet was frozen at -20°C. Periplasmic lysis was carried out using 10 mL of RIPA buffer (Sigma #R0278) for each pellet from 500 mL of bacterial culture, supplemented with complete Mini EDTA-Free protease inhibitor cocktail (Roche #11836170001). After incubation with rotation for 6-9 hours, the lysate was centrifuged at high speed (15000 g for 30 minutes at 4°C) to remove cell debris. The clarified supernatant was filtered through a 0.2 pm syringe filter and purified using Akta Pure FPLC (Cytiva).

[0288] For the first purification step, HiTrap Chelating 5 mL columns (Cytiva #17040903) were used for ion metal affinity chromatography (IMAC) purification (Nickel charged). The working buffer was 20 mM HEPES, 0.5 M NaCI, pH 7.5, and typically, sdAbs were eluted in the range of 100-300 mM imidazole (Sigma #56750). A second purification step included ion exchange chromatography (I EC) in RESOURCE Q / S columns (Cytiva #17117701 #17117801), depending on the isoelectric point of each sdAb. For this purpose, IMAC-purified sdAbs were dialyzed in 20 mM HEPES, pH 7.5, without NaCI, using a 6 kDa membrane (Thermo Scientific #88242) overnight at 4°C. Elution of sdAbs varied according to the pl-pH difference and, in all cases, corresponded to the major peak. Finally, purified sdAbs were dialyzed in PBS overnight, and protein concentration was assessed using the Pierce BCA Kit (Thermo Scientific #23225).

[0289] Determination of relative affinities by ELISA

[0290] To determine the EC50 of the ssdAbs, high-binding ELISA plates were coated with 0.25 pg / well of CD33 overnight at 4°C, blocked with 1% BSA in PBS before offering serial dilutions (1 :2) of the starting sdAb concentration of 10 pg / mL in a final volume of 100pL. sdAbs were then incubated for 1 hour at room temperature, followed by the addition of a 1 / 500 dilution of the anti-HA HRP secondary antibody after washing five times with PBS containing 0.05% Tween 20. The plates were further incubated for 1 hour, and HRP activity was revealed using Opti-EIA substrate reagents.

[0291] Determination of kinetic and thermodynamic constants by SPR

[0292] On the Biacore X100 device (Cytiva), an NTA sensor chip (Cytiva #BR100034, Lot.10335787) was charged with Nickel solution and disposed of His-tagged CD33 ectodomain on flow cell 2, resulting in a non-covalent attachment for the protein (C- terminal). Consecutively, EDC / NHS reagents were added to cross-link the CD33- oriented ectodomain covalently to the chip surface, and nickel was stripped with EDTA solution to eliminate non-covalently bound protein. Flow Cell 1 was used as a reference, following the same procedure but without adding CD33. 388 Rlls of CD33 were finally immobilized covalently. The working buffer was HBS-EP+ to avoid metal ions from chelating on the NTA surface, given that sdAbs also have histidine tags. Serial dilutions of the five candidates sdAbs (Nb1 , Nb3, Nb8, Nb16, and Nb17) were made: 1000, 333, 111 , 37.0, 12.3 nM, and a single-cycle kinetics (SCK) protocol was followed. For each sdAb, three startup cycles were performed before four sample cycles: the first and last cycles with 0 mM of sample (baseline controls) and duplicate cycles in the middle with the five concentrations. The sample flow was set at 10 pL / min, with a contact time of 300 s and a dissociation time of 600 s. Chip regeneration was performed using 10 mM glycine at pH 2 three times between cycles (contact 30 s, at 30 mL / min flow). SCK curves were fitted to a 1 :1 binding model to determine the rate constants of association: kon(M'1s_1), and dissociation: kOff (s-1), and the global dissociation constant: KD (KD (M) = k0ff / k0n) that indicates the binding affinity. Fitting was considered good when tc > 100 x ka, II value > 25, and Chi2< 10% Rmax, according to manufacturer instructions. Tc: Flow rateindependent component of the mass transfer constant, ka: Association rate constant (M-1s-1), II value: estimate of the uniqueness of the calculated values for rate constants and Rmax, Chi2: closeness of fit, calculated as the average squared residual, Rmax: Analyte binding capacity of the surface (Rll).

[0293] Epitope binning assay by BLI

[0294] For epitope binning assays, biotinylated CD33 (bio-CD33) was immobilized on Octet® high-precision Streptavidin 2.0 SAX2 sensors (Sartorius #18-5136) in the Octet N1 device at a concentration of 100 nM, resulting in a signal increase of 1.5-2 nm. sdAb concentrations were 400 nM for the first sdAb and 200 nM for the second one in competition experiments. Cycle steps were as follows: i) 30s initial baseline (PBS), ii) 300s of bio-CD33 immobilization, iii) 30s of pre-binding baseline (PBS), iv) 600s of 1stsdAb binding, v) 600s of 2ndsdAb binding. Aligned curves were graphed using GraphPad Prism 8.0. For the interaction matrix representation of the competition, maximum signal (100%) was calculated as the signal obtained by binding the sdAbs to the CD33 immobilized sensor directly (no competitor) and it was graphed in a colour scale reflecting a value of 1 for total non-com petitors and 0 for total competitors. AML cell line staining with CD33 sdAbs

[0295] The MOLM13 and HL60 cells were harvested in U-bottom 96-well plates (100,000 cells / well) and washed with FACS buffer. For reference, the anti-CD33 antibody (clone WM53) BV510 conjugate (BioLegend #303421) was used alongside BV510 Mouse Igd K isotype control (BioLegend #400171) in a 1 / 100 dilution using 50 pL for staining. In other wells, sdAbs (HA-tagged) NB1 , Nb3, Nb8, Nb16 and Nb17 were used as the primary antibody at a concentration of 1 pg / mL using 100 pL per well (100 ng / well) and incubated for 30 minutes at room temperature. An irrelevant sdAb was used as the isotype control in this step. After washing with 200 pL of FACS buffer, a 1 / 100 dilution of anti-HA PE-conjugated antibody was used as the secondary antibody, incubating for 15 minutes at room temperature. All stained cells were transferred to FACS tubes and acquired in Cytoflex Dx to evaluate CD33 expression with both staining methods.

[0296] Second generation CAR design

[0297] The CAR design was based on a second-generation CAR scaffold using the EGF1a promoter, CD8 signal peptide, CD8a hinge and transmembrane domain, 41 BB (CD137) co-stimulus domain, and CD3 signalling domain, in a third-generation self-inactivating lentiviral vector pCCL . Truncated epidermal growth factor receptor (EGFRt) was used as a reporter molecule in most cases, although mCherry and BFP were also used in some instances. sdAb sequences were inserted in the place of the ScFv binding domain in silico. All constructs were synthesized by GenScript.

[0298] Lentivirus generation

[0299] Lentiviral vectors were generated in HEK293T cells using established protocols. In summary, 6x106cells were co-transfected with the pCCL vector harbouring the different CAR constructs and packaging plasmids: pMDLg / pRRE (Gag / Pol), pRSVRev, and pMD2.G (VSVG envelope), using Lipofectamine 2000 (Invitrogen). After 40 hours, supernatants were harvested, filtered, concentrated with Lenti-X Concentrator (TakaraBio #631232), according to the manufacturer's instructions, frozen in liquid nitrogen, and stored at -80°C until required. Titration was performed by infecting HEK 293T cells in 24-well culture plates (100,000 cells / well) with serial dilutions of the concentrated lentivirus and measuring the EGFRt+ (CAR+) population by EGFR-APC staining and flow cytometry.

[0300] Activation profile in Jurkat TRP system Jurkat-TPR cells (kindly provided by P. Steinberg, Medical University of Vienna) were transduced at a multiplicity of infection (MOI) of 1 with the My96 control CAR and the 5 candidate sdAb-based CARs lentiviral vectors. Resulting cells were co-cultured in triplicate alongside MOLM13 (CD33+) and CD33 knockout MOLM13 cells as negative controls. Non-transduced Jurkat-TPR cells served as the control for baseline fluorescence levels. CAR expression was determined using an anti-EGFRt-APC (allophycocyanin) antibody (clone AY13, BioLegend). Activation of the NFAT, NF-KB, and AP1 pathways was assessed before and 24 hours after co-culture with tumor cells by measuring enhanced GFP (eGFP), enhanced cyan fluorescent protein (eCFP), and mCherry emissions, respectively, using the CytoFLEX LX Flow Cytometer (Beckman Coulter).

[0301] The designed second-generation sdAb-CAR constructs, featuring 4-1 BB as the costimulatory domain and truncated epidermal growth factor receptor (EGFRt) as the reporter, were initially assessed in this triple parameter reporter (TPR) system (Rosskopf S, et al., Oncotarget. 2018 (25): 17608-19) which facilitates the report of the CAR based on three main activation signalling pathways: nuclear factor of activated T cells (NFAT), nuclear factor kappa B (NF-KB), and activator protein 1 (AP1) by producing green fluorescent protein (GFP), blue fluorescent protein (BFP), or mCherry (mCh), respectively. Co-cultivation of these cells with tumor cell lines facilitates the evaluation of the activation profile via flow cytometry. The CAR-mediated activation of these transcription factors in the absence of tumoral cell antigenic stimulation is known as tonic signalling.

[0302] CAR-T cell production

[0303] Peripheral blood samples of 100-150 mL were extracted from healthy donors aged between 18-28 years (male and female) listed at the Clinica Universidad de Navarra as blood and bone marrow donors. Blood with EDTA as a coagulant was diluted at a ratio of 2:3 blood to PBS. Subsequently, 25 mL of diluted blood was carefully layered on 15 mL of Ficoll in 50 mL centrifugation tubes for density gradient centrifugation (400g, 30 min with no acceleration or deceleration). Peripheral blood mononuclear cells (PBMCs) were collected and washed several times with PBS. After counting using Nexcelom chambers (Revvity #CHT4-SD100) and Cellometer® K2 (Nexcelom, Revvity), magnetic anti-CD4 and anti-CD8 Microbeads (Miltenyi #130-045-101 #130-045-201) were added (6 pL of each per million cells) and incubated for 20 min at 4°C in a volume of 1 mL per 100 million PBMCs. Magnetic cell sorting was performed using the autoMACS Pro Separator (Miltenyi Biotec) with the Possel program. The positive fraction containing T cells (CD4+ and CD8+) was then activated using T cell TransAct (Miltenyi #130-111- 160) CD3 and CD28 agonist beads and cultured at a concentration of 1 million cells / mL in RPMI medium supplemented with 3% human serum (Sigma #H4522), 1% penicillin / streptomycin, 625 lll / mL IL7, and 85 lll / mL IL15 to maintain the stem-like phenotype.

[0304] 48 hours later, T cells were infected with lentivirus at a MOI of 3 with 10 pL / mL of LentiBOOST (Sirion Biotech). On day 5, cells were centrifuged to eliminate the TransAct and lentivirus remains, and resuspended in new RPMI complete medium with cytokines to a concentration of 1 million cells per mL. Culture was maintained until day 12-14, with cell concentration maintained at 1 million cells / mL every 2-3 days. Counts were recorded to evaluate T-cell doublings. The percentage of CAR expression was assessed on day 7-9 of production by staining 200,000 cells with APC-antiEGFRt antibody and performing flow cytometry analysis using Cytoflex Dx.

[0305] CAR-T phenotypes in flow cytometry

[0306] The initial T-cell and final CAR-T cell (day 12-14) subpopulations, activation, and exhaustion phenotypes were characterized using five different panels detailed in the Supplementary Material. Subpopulation panels included chemokine receptors and surface markers CCR7, CD45RA, CXCR3, CD4, and CD8. Activation markers included CD69, ICOS, HLA-DR, and CD123, while exhaustion markers included LAG3, PD1 , TIGIT, and TIM3. Both activation and exhaustion panels had their corresponding isotype controls. In all phenotype panels, CD3, CD8, and the truncated EGFR (EGFRt) as a reporter of CAR expression were included. Beriglobin at a concentration of 1 pg / mL was used as an IgG blocking agent, and FACS buffer (PBS, 1% BSA, 0.02% EDTA) was used as a diluent. Samples were acquired using a FACS Canto device.

[0307] Cytotoxicity assay and cytokine production

[0308] To assess CAR-T cell cytotoxicity, GFPLuc+ MOLM13, MV411 , and HL60 cell lines were used as AML target tumoral cells. Co-cultures were set in a 96-well Il-bottom culture plate using 10,000 tumoral cells and a starting effectortumor ratio of 3:1 , followed by ratios of 1 :1 , 1 :0.3, and 1 :0.1 (30,000, 10,000, 3,000, and 1 ,000 CAR-T cells, respectively) in complete RPMI medium. After 24 hours of incubation at 37°C with 5% CO2, cells were centrifuged at 600g for 3 minutes, and the supernatant was collected for cytokine assessment. Pellets containing CAR-T cells and potentially lysed tumoral cells were washed with PBS, resuspended in 30 pL of fresh RPMI medium, and transferred to a NUNC white flat-bottom plate. Then, 30 pL of brightGlo reagent was added to each well, and after 5 minutes, bioluminescence was measured using a GloMax Discover device (Promega).

[0309] IFN-y, TNF-a, and IL-2 concentrations in the co-culture supernatants (1 :1 and 1 :0.3 ratios) from the cytotoxicity tests were assessed using OptiEIA™ ELISA kits (BD #555142, #555190, #555212) following the provider's instructions. Briefly, plates were coated with diluted capture antibody for 24-48 hours at 4°C and then blocked with assay diluent. Different dilutions of the samples and the standard curve were incubated for 1 hour at room temperature. After washing, HRP-Streptavidin pre-conjugated detecting antibodies were incubated for 2 hours at room temperature and washed 10 times. HRP activity was detected with OptiEIA™ substrate reagents, and absorbance at 450 and 565 nm was measured using a GloMAX Discover device. Standard curves were constructed, and sample absorbance values were interpolated to determine cytokine concentrations.

[0310] To ensure specificity, CD33 negative MM1S (RRID:CVCL_8792) was used as control cell line to perform cytotoxicity and cytokine production assays, following the aforementioned procedures (Figure 9).

[0311] Repeated stimulation of CAR-T cells

[0312] For continued re-stimulation, after expansion (12-14 days), 4 million CAR-Ts were cocultured at a 1 :1 EffectorTarget (E:T) ratio with MOLM13 cells, totaling four challenges over 2 weeks (2 challenges per week, separated by 2-3 days). Cell counting was performed using cell counter chambers (Nexcelom #CHT4-SD100-014) and Cellometer K2 fluorescent cell counter (Nexcelom) and %CAR was monitored by EGFRt staining and flow cytometry on DxFIex flow cytometer (Beckman Coulter). Based on cell count and %CAR, MOLM13 cells were added to achieve the corresponding number of CAR+ cells. The culture media consisted of RPMI 3%HS 1%Penicillin / Streptomycin without added cytokines. New cytotoxicity assays and final phenotype stainings for flow cytometry were performed after the 2 weeks of re-stimulation.

[0313] In Vivo MOLM13 NSG Mouse Xenograft Model

[0314] To evaluate the preclinical activity of the sdAb-based CAR-Ts, NSG mice (Rosskopf S, et al., Oncotarget. 2018 (25): 17608-19) aged between 10-12 weeks were xenografted with 50,000 MOLM13 (CD33+, luciferase+) cells via intravenous tail injection. Three days after tumor injection, mice were treated with high (3 million), medium (1.5 million), and lower (0.5 million) doses of the different CAR-T cells, untransduced T cells (UTD) from the same human donor, or PBS as a control. Animals showing loss of mobility, high tumoral mass, or noticeable pain were immediately sacrificed to prevent animal suffering. Survival was calculated with Day 0 being the day the tumor was injected. All experimental protocols received approval from the Ethics Committee of the University of Navarra and the Institute of Public Health of Navarra in compliance with European Council Guidelines. NSG mice were obtained from The Jackson Laboratory (JAX), bred, and housed in a pathogen-free facility within our institution.

[0315] For tumoral progression measurement in a separate assay, luciferase activity was measured in IVIS® Spectrum imaging device (Revvity). Animals were injected intraperitoneally with 100 pL of luciferin solution #1-360222-200 (Regis Technologies) and anesthetized with an inhalation mixture of isoflurane and oxygen. 5-10 minutes after luciferin injection, images were captured and saved for further analysis. Images were adjusted to the same scale and radiance values of each ROI were averaged and graphed.

[0316] In vivo evaluation of CAR-T cell phenotype and expansion

[0317] To assess the in vivo expansion, persistence, activation, and exhaustion profiles of CAR- T cells, an experiment was conducted using the previously described xenograft model. in NSG mice (Figure 7). Mice were intravenously injected with 5*104MOLM13 cells and treated 48 hours later with 1.5x106CAR-T cells per animal. Experimental groups received CAR-T cells expressing different antigen recognition domains: conventional ScFv, Nb3, or Nb16-based CARs. To investigate CAR-T cell expansion and phenotype over time, a representative subset of animals (two males and two females per group) was euthanized on days 5 and 7 post-tumor inoculation, corresponding to days 2 and 5 after CAR-T cell administration.

[0318] Spleens were harvested and mechanically dissociated to obtain single-cell suspensions. Red blood cell lysis was performed using ACK lysis buffer (Gibco™ A1049201), followed by centrifugation and washing. For flow cytometry analysis, two antibody panels were used. Panel 1 was designed to assess expansion / persistence and included staining for CD3 and EGFR (as a marker of CAR expression). Panel 2 targeted activation and exhaustion markers (TIM-3, HLA-DR, and 4-1 BB), and additionally included CD4, CD8, CCR7, CD45RA, and EGFR to enable precise identification and phenotypic characterization of CAR-T cell subsets. Evaluation of co-stimulatory CAR domains

[0319] Nb16-based CAR-T cells targeting CD33 and incorporating either CD28 or 4-1 BB as costimulatory domains (Nb16-CD28 and Nb16-4-1 BB< were generated and evaluated for cytotoxic activity, cytokine production in vitro, and antitumor efficacy in vivo using the standard MOLM13 AML xenograft model (Figure 8). In vitro cytotoxicity assays were performed by co-culturing CAR-T cells with MOLM13-luciferase+ target cells at varying effector-to-target ratios for 24-72 hours, and cytotoxicity was quantified by measuring luciferase activity as a proxy for tumor cell viability. Cytokine release (IFN-y and IL-2) was measured from supernatants using ELISA after 24 hours of co-culture. For in vivo studies, NSG mice were intravenously injected with 5x104MOLM 13 cells and treated 48 hours later with 1.5x106CAR-T cells. Survival was monitored over time to compare the therapeutic impact of each CAR design.

[0320] Allogenic CAR-T cells based on SdAbs against CD33

[0321] For the production of genetically modified CAR-T cells expressing a chimeric antigen receptor (CAR) and edited to silence HLA and TCR expression, an optimized procedure is followed (Figure 10 A). CD4+CD8+T cells are isolated through magnetic selection. The selected cells are adjusted to a concentration of 1 x10® cells / mL and activated for 48 hours in serum-free TexMACS medium supplemented with 10 pL / mL of TransAct (Miltenyi Biotec), 625 IIJ / mL of recombinant human IL-7, and 87.5 IIJ / mL of recombinant human IL-15 (Miltenyi Biotec). For large-scale expansion using GMP-grade reagents, 2 mL of TransAct-GMP per 100x10® cells is used. On day 2 (post-activation), the cells undergo electroporation for dual genetic modification: to introduce CAR expression and to disrupt HLA and TCR expression. This is achieved by electroporating 0.5 pg / 106cells of minicircle (MC) DNA encoding the Nb16-based CAR (MC-nano), 0.25 pg / 106cells of SB100X mRNA, and a preassembled RNP complex composed of 3 pM sgRNAs targeting TRAC and B2M loci and 1 .5 pM Cas9 protein. Following electroporation, the cells are cultured for 12 days in serum-free TexMACS medium supplemented with 625 IIJ / mL IL-7 and 87.5 IIJ / mL IL-15. On day 7 (i.e., five days post-electroporation), additional supplementation with 625 IIJ / mL IL-7 and 87.5 IIJ / mL IL-15 is performed. On day 14, the culture is terminated, and TCR+cells are depleted. Functional and phenotypic analyses are subsequently conducted on the resulting cell population. Using this protocol, large-scale manufacturing has been successfully achieved, validating the procedure previously optimized at small scale. The resulting cells have been employed in in vivo efficacy studies of CD33-targeting CAR-T cells, which were genetically engineered to express a CAR derived from a single-domain antibody using the Sleeping Beauty transposon system (MC-nano and SB100X mRNA), and CRISPR-Cas9 technology to silence endogenous HLA and TCR expression.

[0322] Statistical Analysis

[0323] Statistical analyses were conducted using GraphPad Prism 8.0. Specific tests utilized in this study are specified in the legend of each Figure.

[0324] RESULTS

[0325] Identification and biophysical characterization of CD33 specific sdAbs

[0326] A phage display sdAb library was generated from immunized llama N°124. Llama's humoral response against CD33 was sufficiently heightened for library construction, reaching an EC50 of 108591 in anti-CD33 ELISA titration (Figure 1 A). The library exhibited a diversity of 1.75 x 1O10ufc (total transformants) and phage titers exceeding 1013. After three rounds of panning, phage enrichment was achieved, as depicted in Figure 1 (B, C, D), illustrating an increase in the O / l ratio (B), heightened signal in M13 polyclonal phage ELISA (C), and a substantial representation of positive clones in preliminary monoclonal screening (D). The amplified phagemids from the final panning round were subcloned into the expression vector and transformed into E. coli BL21. From twenty isolated colonies included in ELISA screening tests (Figure 1 E), 18 clones were positive (90%) against CD33 and were subsequently sequenced. Out of the identified sequences, five were significantly different: Nb1 (also referred to as CD33_SDAB1 or sdAbl), Nb3 (also referred to as CD33_SDAB3 or sdAb3), Nb8 (also referred to as CD33_SDAB8 or sdAb 8), Nb16 (also referred to as CD33_SDAB16 or sdAb16), and Nb17 (also referred to as CD33_SDAB17 or sdAb17). The phylogenetic tree, considering the mean distance between sequences, delineates five clustered families, with each of the aforementioned sdAbs representing one of these families (Figure 1 F). Sequence dissimilarities may indeed indicate diverse binding epitopes and affinities, thereby increasing the likelihood of obtaining sdAbs with varied properties. The five sdAbs were produced and purified successfully, showing excellent purity and activity (Figure 1 G-H).

[0327] To characterize the binding affinity and describe the binding kinetics for each sdAb, ELISA end SPR were performed. The five CD33 sdAb candidates exhibited varying EC50 values in ELISA (Figure 2A), corresponding to their relative affinities (data shown in Table 1). Binding kinetics experiments conducted via SPR (SOK curves displayed in Figure 2C) facilitated the determination of kon, koff, and KD, which are also detailed in Table 1. The affinities assessed by SPR correlate with the EC50 differences observed in ELISA. Among the candidates, Nb16 displayed the highest affinity (KD=3.9nM), while Nb3 exhibited the lowest affinity (KD=115nM).

[0328] Tablel : CD33 binding affinity and kinetic constants for the five sdAb candidates and the reference scFv.

[0329] Antibody EC50(M) KD(M) kon(1 / M*s) koff (1 / s)

[0330] Nb1 6.40E-09 2.99E-08 3.68E+04 1.10E-03

[0331] Nb17 4.05E-09 4.99E-09 1.65E+04 8.24E-05

[0332] ScFv 8.99E-11 3.52E-10 5.12E-05 1.80E-04

[0333] “My96”

[0334] To determine if sdAbs were able to bind to CD33 naturally expressed on the surface of AML cells, besides recombinant CD33 ectodomain, flow cytometry binding assay was performed. As illustrated in Figure 2B, identified sdAbs can also bind to CD33 expressed on the surface of AML cell lines, exhibiting a CD33-dependent pattern akin to that of the anti-CD33 mouse monoclonal antibody (clone WM53) in flow cytometry. Nb3 showed poor binding probably due to its low affinity, which probably unmatched the conditions of the staining assay for flow cytometry.

[0335] To explore if these antibodies bind to similar epitopes of CD33, BLI epitope binning was performed using the three sdAb candidates that exhibited the least dissociation (lower koff) to the biotinylated CD33 immobilized in the streptavidin sensor SAX2: Nb1 , Nb8, and Nb16, as the initial-binding antibodies in an in-tandem approach, with one control sensor lacking primary antibody in each case. The five sdAb candidates were utilized as secondary antibodies in all instances, and the resulting binding curves are depicted (Figure 2D). All assessed sdAbs exhibited partial or complete competition for antigen binding, as summarized in the interaction matrix shown in the right panel of Figure 2D, suggesting epitope overlapping, steric hindrances, or conformational changes in the antigen that impede dual binding. Binding of Nb1 and Nb16 completely precludes the binding of Nb3 and Nb17, indicating epitope overlapping. Partial competition is observed between Nb8 and Nb1 (both ways) or Nb8 and Nb16 (only when Nb8 binds first). These observations are taken into consideration for experimental design and combined approaches.

[0336] To establish the inventive contribution of the CD33-targeting sdAb-based CAR constructs, comparative experiments were conducted against the well-characterized anti-CD33 scFv My96, including affinity measurements and epitope binning assays to confirm distinct binding properties. Affinities of all the selected sdAbs, KD ranging from 4.18 - 115.00 nM, were different from the scFv fragment corresponding to the reference CAR binding moiety “My96”, with a KD of 0,352 nM as determined by SPR.

[0337] Generation and selection of VHH-based CAR constructs targeting CD33

[0338] To characterize the selected sdAbs as binding motifs in designed 2ndgeneration CAR constructs, represented in Figure 2E, activation assays of Jurkat triple parameter reporter (TRP) cells were performed. Jurkat cells were infected with the same moiety of infection (MOI) of the different lentiviral vectors harboring each sdAb-based CAR construct and ScFv. based reference CAR. Expression of EGFRt, used as reporter of the CAR expression, was evenly distributed among all construct as observed in flow cytometry (Figure 2F), denoting equal CAR expression, thus CAR density does not influence the observed activation profiles. In Figure 2G, NFAT, AP1 , and NF-KB tonic signals are depicted in green bars for both the My96 control CAR and the five sdAb- based CARs evaluated. Generally, sdAb-based CARs exhibited less tonic signal than the control My96 CAR, except for the Nb8 CAR, which displayed a similar percentage of NFAT-activated cells as My96 and even higher levels of NF-KB and AP1 tonic signal. Activation of CARs over CD33+ MOLM13 cells was successfully achieved by all CAR constructs, with no significant differences observed (Figure 2G). However, CAR activation on CD33 knockout MOLM13 cells was consistently lower in all cases, although Nb16 and Nb17 exhibited significantly lower activation, likely due to their higher antigen specificity. Subsequent to these findings, three sdAb CARs were selected for further evaluation in human T cells, both in vitro and in vivo. These candidates, all displaying low tonic signaling and off-target activation, were as follows: Nb16, with the highest binding affinity; Nb3, with the lowest binding affinity; and Nb1 , with intermediate binding affinity. sdAb-CARTs against CD33 exhibit cytotoxicity and proinflammatory cytokine production in vitro To characterize sdAb-based CD33 CAR-T cells, phenotypically and functionally, a total of 10 CAR-T productions from young healthy peripheral blood donors were conducted. Overall, CAR expansion was successful in all cases, with slightly higher proliferation observed in some sdAb CARTs compared to My96 CAR-T (Figure 3A). These differences may relate to reduced CAR-induced toxicities, decreased tonic signaling, or the effect of lentiviral infection volumes. After 12-14 days of expansion, CAR-T cells were characterized both phenotypically, via subpopulation, activation, and exhaustion marker staining using flow cytometry, and functionally, by determining CAR-T cytotoxicity and assessing cytokine production (IFN-y, IL-2, and TNF). CAR-T phenotypes closely resembled those of My96 CAR-T in all cases (Figure 3 B-D).

[0339] T cells comprised, on average, 75-80% CD4+ cells and 20-25% CD8+ T cells. Subpopulations of both CD4+ and CD8+ T cells were primarily composed of effector memory cells, followed by central memory cells, effector cells, with very few stem cell memory cells compared to untransduced T cells. Naive T cells were highly represented only in the basal phenotype (day 0 before activation and expansion). The activation marker ICOS was expressed in >90% of all CD4+ and CD8+ CAR-T cells, while HLADR and CD69 expression varied between 10-40% in all CAR-T cells and untransduced T cells. Very low percentages of T cells expressed the intrinsic 4-1 BB activation marker. Exhaustion markers LAG3, PD1 , and TIGIT were minimally expressed, although TIM3 was expressed in 60-70% of CD4+ T cells and 80-90% of CD8+ T cells.

[0340] CAR-T cytotoxicity was observed across three AML cell lines: HL60, MOLM13, and MV411 , with decreasing expression levels of CD33 in the same order (Figure 3E). No significant differences were observed in cytotoxicities between My96 and sdAb CARTs, indicating similar levels of tumor killing at all effector-to-target (E:T) ratios. Cytotoxicity was also evaluated on CD33 knockout MOLM13 cells, demonstrating significantly lower toxicities. Nb16 exhibited the least off-target toxicity. The concentrations of CAR-T cytokines in the supernatants of CAR-T co-cultures (1 :1 E:T ratio) with the AML cell lines are depicted in Figure 3C. Notably, sdAb CAR-Ts exhibited significantly higher IL-2 and IFN-y production than My96 (Figure 3F), which may pose a disadvantage in terms of associated toxicities but can promote T cell proliferation and tumor killing.

[0341] Cytotoxicity and cytokine production from ScFv-, Nb3- and Nb16-CAR-T cells were also assessed in co-culture with the control MM1S (CD33-) cell line as a measure of specificity, resulting in poor toxicities against MM1S cells, very low secretion of IFN-y and undetectable levels of IL2 (Figure 9). sdAb-CARTs against CD33 maintain cytotoxic activity after repeated stimulation with tumoral cells

[0342] To evaluate the long-term functionality of the sdAb-based CAR-T cells, repeated stimulation assay was conducted. When exposed to tumoral cells, CAR+ T cells proliferated very similarly, with Nb16 CAR-Ts demonstrating slightly better proliferation, as shown in the population doubling graph in Figure 4A. After continued exposure to tumoral cells (MOLM13) for 2 weeks, T cells were tested in vitro to assess their cytotoxicity, cytokine production, and phenotypes once again.

[0343] Cytotoxicity remained consistently high in all cases, comparable to the first exposure (Figure 4E), with no significant differences between My96 CAR-Ts and the Nb16 and Nb3 CAR-Ts at high E:T ratios. However, at 0.1 :1 E:T ratios, Nb16 exhibited significantly higher cytotoxicity. Cytokine production differed significantly between My96 CAR-Ts and sdAb-based CAR-Ts, consistent with the first exposure (Figure 4F). All stimulated sdAb CAR-T cells exhibited significantly higher expression of IL2, while only Nb16 showed significantly higher IFNy levels.

[0344] In general, final T cells were >90% CAR+, with CAR-Ts comprising around 60% CD4+ and 40% CD8+ (Figure 4C). Both subpopulation phenotypes showed higher proportions (60-80%) of central memory T cells (Tern) and 10-40% of effector T cells (Te) for both CD4 and CD8 populations, as depicted in Figure 4B. Very low percentages (<5%) of central memory and stem cell memory cells were detected, and naive T cells were absent in the restimulated samples.

[0345] As shown in Figure 4D, the activation marker ICOS was expressed in all stimulated CARTs, with a high percentage of positive cells ranging from 80-100%. HLADR expression was also high but was higher in My96 than in sdAb-based CAR-Ts. CD69 and 4-1 BB were expressed on all stimulated CAR-Ts, although at lower percentages of positive cells. Exhaustion marker TIM3 was highly expressed in all stimulated CAR-Ts but slightly higher in sdAb CAR-Ts, particularly in Nb16 CD4+ CAR-T cells. This increased TIM3 expression, though unexpected, may be attributed to the lower expression of other exhaustion markers, explaining the increased proliferation and cytotoxicity at low E:T ratios of Nb16 CAR-Ts. TIGIT expressing cells were detected in all cases but were slightly lower in Nb16 CAR-Ts. PD1 expression was observed only in stimulated Nb1 CARTs, but no statistical significance was obtained. Very low levels of LAG3 were detected in CD8 CAR-T cells. Overall, CAR-T phenotypes were very similar between groups, with the main observed differences being the increased IL2 and IFN production, especially by Nb16 CAR, which also exhibited a better proliferation profile and higher cytotoxicity at low E:T ratios.

[0346] In vivo experiments demonstrate sdAb-CART high efficacy in xenograft AML mouse model

[0347] To evaluate the in vivo functionality of the sdAb-based CAR-T cells, we evaluated survival and tumor progression in AML xenograft NGS mouse model. Three CAR-T cell doses were evaluated, in a stress test. Survival curves for each dose are shown in figure 5. At high dose (3 million CARTs per mouse), significant increases were observed in mouse survival across all CAR-T treatments compared to control groups receiving PBS or untransduced (CAR-) T cells from the same donor (Figure 5C) Although no significant differences were observed between treatment groups, 20% of mice in the Nb16 group survived at day 70, whereas all mice in the other treatment groups had perished. This observation suggested a possible improvement in CAR-T efficacy that was not evident at this high dose. At lower doses:1.5 and 0.5 million CAR-Ts per mouse, some CAR-T cells lose effect, while others maintain it. The dose of 0.5 million CAR-Ts (Figure 5A) resulted in much lower survival across all treatment groups, but fortunately, the dose of 1.5 million CAR-Ts (Figure 5B) showed decreased survival in the My96 group while maintaining the survival profile in the Nb3 and Nb16 groups. This indicated significant differences between these groups, suggesting that sdAb CAR-Ts might exhibit higher efficacy in this AML model. Nb16 demonstrated the best performance in this model, and while the mechanisms underlying this improvement are not clear, potential factors include higher IL2 and IFNy production, increased proliferation after stimulation, and higher cytotoxicity at low CAR-T concentrations compared to My96. Tumor progression assay corresponded with the observations in survival assays, although in this case sample size was smaller. Tumor progressed significantly less in all CAR-T cell treated mice, in comparison with UTD T cell treated mice. Between CAR-T cell treatments, tumoral cells progressed slightly more in My96 CAR-T cell group, especially in the last periods of time (Figure 5D-E). All these findings suggest that sdAb-based CAR-T cells, notably Nb16, are very promising therapeutic agents against AML.

[0348] Nb16, as the final candidate, was further tested in vivo, particularly comparing costimulatory signaling domains and evaluating combinatorial approaches. To ensure that the 41 BB co-stimulatory signal was the most appropriate, Nb16 CAR constructs harboring CD28 stimulus domains ( Nb1628z) were also evaluated alongside the original Nb16 41 BB (Nb16BBz) CAR in another in vivo experiment (Figure 8). As expected, the 41 BB construct seemed to outperform the CD28 construct, correlating with other studies of CARTs against CD33 in AML models. This may be related to the improved mechanisms of 41 BB signaling, making CAR-Ts more effective in long-term responses. To investigate if combinations of these sdAb CARs exhibit therapeutic synergy, two combination approaches were tested: (i) using a 1 :1 mixture of Nb16 CARTs and Nb3 CAR-Ts (competitors with different affinities) and (ii) co-transfecting Nb16 and Nb1 (partial competitors with similar affinities) in the same T cells. Both tested combinations did not show a significant increase over Nb16 treatment alone, although T cells co-transduced with Nb16 and Nb1 CARs presented an attractive survival profile.

[0349] Furthermore, the in vivo characterization of CAR-T cells included the evaluation of their immunophenotypic profile, proliferation, and persistence in treated animals, as detailed in the Materials and Methods (Figure 7). These experiments demonstrated that Nb16- CAR-T cells exhibit markedly higher IL-2 production, a feature directly associated with enhanced in vivo proliferation and sustained activity. As a result, Nb16-CAR-T cells achieve superior antitumor efficacy at lower cell doses, highlighting a significant technical advantage over existing CD33-targeting approaches. No substantial differences were observed in the immunophenotypic markers of CAR-T cells isolated from treated mice at early (day 3) and later (day 7) timepoints post-administration. However, a marked increase in the number of CAR-T cells was detected on day 7 in animals receiving Nb16- and Nb3-based CAR-T cells, indicating enhanced proliferative capacity and persistence of these constructs in vivo.

[0350] Allogenic CAR-T cells based on Nb16 were functional against AML cells

[0351] Using the previously established conditions, a minicircle DNA construct encoding a CD33-targeting CAR based on a single-domain antibody (MC-nano) was evaluated. (Figure 10). Three experimental conditions were compared across three independent experiments:

[0352] • Condition 1 (AL-NB): Allogeneic CAR-T cells expressing the sdAb-based CAR (MC-nano) and edited to silence both HLA and TCR.

[0353] • Condition 2 (AL-MC): Allogeneic CAR-T cells expressing a conventional scFv- based CAR and edited to silence HLA and TCR.

[0354] • Condition 3 (WT-NB): Non-edited CAR-T cells expressing the sdAb-based CAR (MC-nano).

[0355] • Condition 4 (UTD): Untransduced, non-edited T cells (negative control). The results demonstrated that CAR expression, as measured by EGFR surrogate marker, was comparable among the three CAR-expressing groups (AL-NB, AL-MC, and WT-NB). Likewise, both allogeneic conditions (AL-NB and AL-MC) achieved similar efficiency in gene editing, as reflected by the reduction in TCR and HLA expression and the generation of a TCR7HLA- population prior to depletion. Following depletion of residual TCR+cells, a comparable enrichment of the double-negative population was observed in both edited groups.

[0356] Cell expansion was also similar between AL-NB and AL-MC groups, indicating that the use of the sdAb-based CAR construct does not negatively impact cell proliferation. Furthermore, all CAR-expressing cell products (AL-NB, AL-MC, and WT-NB) exhibited equivalent cytotoxic capacity in vitro against MOLM 13 target cells expressing CD33.

[0357] These results collectively confirm that the MC-nano construct enables efficient CAR expression, supports successful genome editing to silence HLA and TCR, and produces CAR-T cells with functional cytotoxic activity against CD33+target cells.

[0358] CDR-grafting of Nb16 against CD33

[0359] The CDRs identified for Nb16 (SEQ ID NO: 18, 19 and 20) were grafted in the corresponding order (FR1 [SEQ ID NO: 62]-CDR1 [SEQ ID NO: 18J-FR2 [SEQ ID NO: 63]-CDR2[SEQ ID NO: 19J-FR3 [SEQ ID NO: 32]-CDR3 [SEQ ID NO: 20]-FR4 [SEQ ID NO: 25]) into consensus sdAb framework sequences obtained from AugurLIama Database, which matches 100% with the consensus framework regions obtained from a smaller internal sdAb database. The grafted sdAb has the sequence according to SEQ ID NO: 64.

[0360] The construct was cloned and produced in parallel with original Nb16, using the exact expression system and purification methods. ELISA was performed by coating high- binding 96 well plates with 0.25 ug of recombinant human CD33 protein (SinoBiological #12238-H08H) at 4°C overnight followed by blocking 1h at RT with PBS BSA 5% and incubating with 1 serial dilutions of the sdAbs starting from a 10 ug / mL solution 1h at RT. Anti-HA-HRP secondary antibody was incubated 1h at RT after 5 washes with PBS then 0.05%. ELISA was revealed after several washes with PBS tween 0.05% using TMB reagents and 450nm absorbance was read in a plate-spectrophotometer. Grafted Nb16 maintained the original functionality, binding to CD33 with very similar relative affinity in comparison with original Nb16 (Nb16 EC50: 26.0 ng / mL and Grafted Nb16 EC50: 78.4 ng / mL) (Figure 6). This observation demonstrates that CDRs alone, are responsible for CD33 binding and thus, the sdAb’s functionality. Constructs that graft these CDRs into structurally similar domains, are likely to maintain the functionality. These Include VH domains of all vertebrate immunoglobulins, and proteins that have an Ig-like structure.

Claims

1. 73CLAIMS1. A single domain antibody (sdAb) that specifically binds to CD33, wherein:(a) the CDR1, CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 18, 19 and 20 or a functionally equivalent variant of one or more of the above;(b) the CDR1, CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 6, 7 and 8 or a functionally equivalent variant of one or more of the above;(c) the CDR1, CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 9, 10 and 11 or a functionally equivalent variant of one or more of the above;(d) the CDR1, CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 12, 13 and 14 or a functionally equivalent variant of one or more of the above; or(e) the CDR1, CDR2 and CDR3 regions comprise respectively the sequences of SEQ ID NO: 9, 13 and 17 or a functionally equivalent variant of one or more of the above.

2. The sdAb according to claim 1 wherein:(a) the FR1, FR2, FR3 and FR4 regions in the sdAb as defined in claim 1(a) comprise respectively the sequences of SEQ ID NO: 33, 34, 35 and 25 or a functionally equivalent variant or one or more of the above;(b) the FR1, FR2, FR3 and FR4 regions in the sdAb as defined in claim 1(b) comprise respectively the sequences of SEQ ID NO: 22, 23, 24 and 25 or a functionally equivalent variants of one or more of the above;(c) the FR1, FR2, FR3 and FR4 regions in the sdAb as defined in claim 1(c) comprise respectively the sequences of SEQ ID NO: 26, 23, 27 and 25 or a functionally equivalent variant of one or more of the above;(d) the FR1, FR2, FR3 and FR4 regions in the sdAb as defined in claim 1(d) comprise respectively the sequences of SEQ ID NO: 28, 29, 30 and 25 or a functionally equivalent variant of one or more of the above; or(e) the FR1, FR2, FR3 and FR4 regions in the sdAb as defined in claim 1(e) comprise respectively the sequences of SEQ ID NO: 31, 23, 32 and 25 or a functionally equivalent variant of one or more of the above.

743. The sdAb according to claim 1 or 2 wherein the functionally equivalent variant of SEQ ID NO: 20 is SEQ ID NO: 21.

4. The sdAb according to claim 3 wherein the sdAb contains the CDR1 , CDR2 and CDR3 according to the sequences SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 21, respectively.

5. The sdAb according to claim 2 wherein the functionally equivalent variant of SEQ ID NO: 33 is SEQ ID NO: 62 and / or wherein the functionally equivalent variant of SEQ ID NO: 34 is SEQ ID NO: 36 or SEQ ID NO: 63.

6. The sdAb according to any of claims 1 to 5 wherein:(a) the sdAb according to claim 1(a) comprises a sequence according to SEQ ID NO: 5 or a functionally equivalent variant thereof;(b) the sdAb according to claim 1(b) comprises a sequence according to SEQ ID NO: 1 or a functionally equivalent variant thereof;(c) the sdAb according to claim 1(c) comprises a sequence according to SEQ ID NO: 2 or a functionally equivalent variant thereof;(d) the sdAb according to claim 1(d) comprises a sequence according to SEQ ID NO: 3 or a functionally equivalent variant thereof; or(e) the sdAb according to claim 1(e) comprises a sequence according to SEQ ID NO: 4 or a functionally equivalent variant thereof.

7. The sdAb according to claim 6 wherein the functionally equivalent variant of SEQ ID NO: 5 is SEQ ID NO: 37 or SEQ ID NO: 64.

8. A biparatopic sdAb characterized in that it comprises a first and second sdAb wherein the first and / or second sdAb is a sdAb according to any one of claims 1 to 7.

9. The biparatopic sdAb according to claim 8 wherein the first sdAb and the second sdAb are arranged such that the first sdAb is N-terminally located in relation to the second sdAb or such that the first sdAb is C-terminally located in relation to the second sdAb.

10. The biparatopic sdAb according to any one of claims 8 or 9 further comprising a peptide linker between the first and the second sdAb.7511. The biparatopic sdAb according to claim 10 wherein the flexible peptide linker comprises a sequence selected from the group consisting of SEQ ID NO: 53 (GGGGS) or SEQ ID NO: 54 (GGGGS GGGGS GGGGS).

12. A conjugate comprising:(a) a first domain comprising the sdAb according to any of claims 1 to 7 or the biparatopic sdAb according to any of claims 8 to 11 , and(b) a second active domain, preferably comprising a therapeutic, binding / targeting or marker agent.

13. The conjugate according to claim 12 wherein the conjugate is a fusion protein in which the second domain is a polypeptide fused with the first domain.

14. A polynucleotide encoding the sdAb or the biparatopic sdAb according to any of claims 1 to 11 or the conjugate according to claim 13.

15. The polynucleotide according to claim 14 further comprising a sequence encoding a signal sequence which is fused in frame to the N-terminus of the sdAb, biparatopic sdAb or of the fusion protein.

16. A vector comprising the polynucleotide according to any of claims 14 or 15.

17. A host cell comprising the polynucleotide according to any of claims 14 or 15 or the vector according to claim 16.

18. A polynucleotide encoding a CD33-specific chimeric antigen receptor (CAR) comprising:(a) a sdAb according to any of claims 1 to 7 or the biparatopic sdAb according to any of claims 8 to 11 ;(b) a transmembrane domain; and(c) at least one intracellular signaling domain and / or costimulatory domain.

19. The polynucleotide according to claim 18 wherein the transmembrane domain is selected from the group consisting of the CD4 transmembrane domain, the CD8a transmembrane domain, the CD28 transmembrane domain, the 4-1 BB transmembrane domain, the CTLA4 transmembrane domain, the CD27 transmembrane domain and the CD3 zeta transmembrane domain.7620. The polynucleotide according to claim 29 wherein the transmembrane domain is the CD8a transmembrane domain, preferably comprising a sequence according to SEQ ID NO: 40.

21. The polynucleotide according to any of claims 18 to 20 further comprising a hinge domain between the sdAb and the transmembrane domain.

22. The polynucleotide according to claim 21 , wherein the hinge domain is selected from the group consisting of: CD8a, CD28, and lgG4 hinge domain; preferably, the hinge domain is the CD8a hinge domain, more preferably the hinge domain comprises a sequence according to SEQ ID NO: 39.

23. The polynucleotide according to any of claims 18 to 22, wherein the at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or any combination thereof.

24. The polynucleotide according to claim 23 wherein the at least one intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of 0X40, CD70, CD27, CD28, CD5, ICAM-1 , LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP 12, and 4-1 BB (CD137), or any combination thereof.

25. The polynucleotide according to claim 24 wherein the at least one intracellular signaling domain comprises the intracellular domain of 4-1 BB.

26. The polynucleotide according to claim 25 wherein the intracellular domain of 4-1 BB comprises the sequence of SEQ ID NO: 41 .

27. The polynucleotide according to any of claims 18 to 26 wherein the at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

28. The polynucleotide according to claim 27 wherein the CD3 zeta cytoplasmatic domain comprises the sequence according to SEQ ID NO: 42.

29. The polynucleotide according to claim 27 or 28 wherein the at least one intracellular signaling domain is located N-terminally with respect to the CD3 zeta intracellular domain.7730. The polynucleotide according to claim 29 wherein the hinge domain is the CD8a hinge domain, the transmembrane domain is the CD8a transmembrane domain, the intracellular signaling domain comprises the 4-1 BB intracellular region and the CD3 zeta cytoplasmatic domain.31 . The polynucleotide according to any of claims 18 to 30 wherein the chimeric antigen receptor further comprises a signal peptide located N-terminal to the sdAb or to the biparatopic sdAb.

32. The polynucleotide according to claim 31 wherein the signal peptide is the CD8 signal peptide and / or the GM-CSF receptor A signal peptide, preferably comprising a sequence according to SEQ ID NO: 38 and / or according to SEQ ID NO: 44.

33. The polynucleotide according to any of claims 18 to 32 wherein the chimeric antigen receptor comprises a sequence selected from SEQ ID NO: 46-51 and SEQ ID NO: 65.

34. The polynucleotide according to any of claims 18 to 33 wherein the chimeric antigen receptor further comprises a detection marker at the C-terminus, wherein the detection marker is connected to the chimeric antigen receptor by a self-cleavable protein sequence.

35. The polynucleotide according to claim 34 wherein the detection marker is huEGFRt according to SEQ ID NO: 45 and the self-cleavable protein sequence is the T2A peptide according to sequence SEQ ID NO: 43.

36. A vector comprising a polynucleotide according to any of claims 18 to 35.

37. A cell comprising a polynucleotide as defined in claims 18 to 35, or a vector as defined in claim 36.

38. A cell according to claim 37 characterized in that it expresses the chimeric antigen receptor encoded by the polynucleotide as defined in claims 18 to 35.

39. The cell according to claim 38 or 39 wherein said cell is selected from a group consisting of an immune cell, a somatic cell and a progenitor cell.7840. The cell according to claim 39, wherein the immune cell is selected from a group consisting of a T cell, a B cell, a NK cell, a neutrophil, a eosinophil, a basophil, a monocyte, a macrophage, a dendritic cell and a mast cell, or a precursor of any of these cells.

41. The cell according to claim 39, wherein said cell is selected from a T cell and a precursor thereof.

42. The cell according to claim 40, wherein said T cell is a primary T cell.

43. The cell according to claim 40, wherein said T cell is a Jurkat T cell.

44. An ex vivo method for obtaining a cell according to any of claims 37 to 43 comprising transducing the cell or precursor thereof with a polynucleotide according to any of claims 18 to 35 or with a vector as defined in claim 36 comprising said polynucleotide.

45. A pharmaceutical composition comprising the sdAb according to any of claims 1 to 7, the biparatopic sdAb according to any of claims 8 to 11 , the conjugate according to claim 12 or 13, the polynucleotide according to any of claims 14 to 15, the vector according to claim 16, the polynucleotide according to any of claims 18 to 35, the vector according to claim 36 and / or the cell according to any of claims 37 to 43.

46. The sdAb according to any of claims 1 to 7, the biparatopic sdAb according to any of claims 8 to 11 , the conjugate according to claim 12 or 13, the polynucleotide according to any of claims 14 to 15, the vector according to claim 16, the polynucleotide according to any of claims 18 to 35, the vector according to claim 36 and / or the cell according to any of claims 37 to 43 for use in medicine.

47. The sdAb according to any of claims 1 to 7, the biparatopic sdAb according to any of claims 8 to 11 , the conjugate according to claim 12 or 13, the polynucleotide according to any of claims 14 to 15, the vector according to claim 16, the polynucleotide according to any of claims 18 to 35, the vector according to claim 36 and / or the cell according to any of claims 37 to 43 for use in the treatment of cancer.

48. The sdAb, biparatopic sdAb conjugate, polynucleotide, vector and / or the cell for use according to claim 47 wherein the cancer is an hematological malignancy.

49. The sdAb, biparatopic sdAb conjugate, polynucleotide, vector and / or the cell for use according to claim 48 wherein the hematological malignancy is characterized by the presence of malignant cells which exhibit CD33 expression on their surface.

50. The sdAb, biparatopic sdAb, conjugate, polynucleotide, vector and / or the cell for use according to claims 48 or 49 wherein said hematological malignancy is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute B cell lymphoblastic leukemia, minimal residual disease (MRD)-positive ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), lymphoplasmacytic lymphoma (LPL), B cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt’s lymphoma (BL), primary mediastinal large B-cell lymphoma (PMBL), marginal zone B cell lymphoma, Hodgkin’s lymphoma (HL), nonHodgkin’s lymphoma (NHL), NK- and T-cell neoplasms, histiocytic neoplasms, mantle cell lymphoma (MCL), hairy cell leukemia (HCL), plasma cell myeloma (PCM), plasma cell leukemia (PCL), and multiple myeloma (MM).51 . A method to detect the presence or level of CD33 in a sample comprising:(a) contacting the sample with the sdAb according to any of claims 1 to 7 or the biparatopic sdAb according to any of claims 8 to 11 or the conjugate according to claim 12 or 13 so as to obtain a mixture wherein said sdAb, biparatopic sdAb or conjugate is allowed to substantially bind with said CD33 when the latter is present in said sample; and(b) detecting the binding of the sdAb or of the biparatopic sdAb or of the conjugate to the CD33 present in the sample thereby providing an indication of the presence or of the level of CD33 in the sample.

52. The method according to claim 51 wherein the sdAb, the biparatopic sdAb or the conjugate comprise a detectable label.

53. The method according to claim 52 wherein the detectable label is selected from an enzyme, a radionuclide, a fluorescent dye, a luminescent substance or biotin.

54. A method for the diagnosis of a disease characterized by an increased level of cells expressing CD33 in a patient, the method comprising detecting the level of cells expressing CD33 in a sample from the patient by a method as defined in any ofclaims 52 to 54 wherein the presence of an increased number of cells expressing CD33 in the sample from the patient with respect to a reference value is indicative of the subject suffering from a disease characterized by an increased level of cells expressing CD33.

55. The method according to claim 54 wherein the disease characterized by an increased level of cells expressing CD33 is acute myeloid leukemia.

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