SINGLE DOMAIN ANTIBODIES AGAINST CLL-1

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. provisional application number

62/774,025 filed on November 30, 2018, and of U.S. provisional application number 62/780,092 filed December 14, 2018, the contents of both of which are herein incorporated by reference in their entirety.

BACKGROUND

[0002] C type Lectin Like molecule 1 (CLL-1, also known as MICL, and Clecl2A) is expressed on acute myeloid leukemia (AML) cells, and on cancer stem cells (CSCs), which are cells that can give rise to additional cancer cells. AML remains a major therapeutic challenge and an unmet need in hematologic oncology. AML is a disease resulting in uncontrollable accumulation of immature myeloid blasts in the bone marrow and peripheral blood, and the disease has multiple subtypes that contribute to the challenge in developing an encompassing targeted therapy. Although there is an increased understanding in the molecular genetics of the disease, there have been relatively few novel therapies approved for AML. Accordingly, there remains a need for novel therapeutics for AML, such as therapeutics that target CLL-1.

SUMMARY OF INVENTION

[0003] In one aspect, the present disclosure provides, among other things, antibodies, or antigen binding fragments thereof, that bind (e.g., selectively bind) CLL-1, compositions useful for binding CLL-1, and methods for treating disease comprising administration of such antibodies, or antigen binding fragments thereof. In some embodiments, the present disclosure provides an antibody, or antigen binding fragment thereof, comprising a VHH described herein. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH having the amino acid sequence of any one of SEQ ID Nos:3-25, or a fragment thereof. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH having a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks one or more of the C-terminal amino acids

T SGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH having a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3- 25, wherein the portion lacks all of the C-terminal amino acids

T SGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH having a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3- 25, wherein the portion lacks one or more of the C-terminal amino acids

T SGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25, and wherein the portion lacks one or more (e.g., 1, 2, 3, 4, 5, or more), additional amino acids. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH having a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3- 25, wherein the portion lacks all of the C-terminal amino acids

T SGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25, and wherein the portion lacks one or more (e.g., 1, 2, 3, 4, 5, or more), additional amino acids. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks one or more of the C-terminal amino acids

T SGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks all of the C-terminal amino acids

T SGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks one or more of the C-terminal amino acids

T SGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25, and wherein the portion lacks one or more (e.g., 1, 2, 3, 4, 5, or more), additional amino acids. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks all of the C-terminal amino acids

T SGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25, and wherein the portion lacks one or more (e.g., 1, 2, 3, 4, 5, or more), additional amino acids. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH

comprising at least one CDR (e.g., CDR1, CDR2, and/or CDR3) depicted in any one of SEQ ID Nos:3-25. In some embodiments, the present disclosure provides an antibody, or antigen binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH comprising at least one CDR that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,

96%, 97%, 98%, 99%, or 100% identical to a CDR (e.g., CDR1, CDR2, and/or CDR3) depicted in any one of SEQ ID Nos:3-25. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH comprising CDR1, CDR2, and/or CDR3 of any one of Groups 1-13 depicted in Table 1A and/or Table IB. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH comprising (i) CDR1 and CDR2; (ii) CDR2 and CDR3; (iii) CDR1 and CDR3; or (iv) CDR1, CDR2, and CDR3 of any one of Groups 1-13 depicted in Table 1 A and/or Table IB. In some embodiments, the present disclosure provides an antibody, or antigen-binding fragment thereof, that binds (e.g., selectively binds) CLL-1, comprising or consisting of a VHH comprising CDR1, CDR2, and CDR3 of Group 1; CDR1, CDR2, and CDR3 of Group 2; CDR1, CDR2, and CDR3 of Group 3; CDR1, CDR2, and CDR3 of Group 4; CDR1, CDR2, and CDR3 of Group 5; CDR1, CDR2, and CDR3 of Group 6; CDR1, CDR2, and CDR3 of Group 7; CDR1, CDR2, and CDR3 of Group 8; CDR1, CDR2, and CDR3 of Group 9; CDR1, CDR2, and CDR3 of Group 10; CDR1, CDR2, and CDR3 of Group 11; CDR1, CDR2, and CDR3 of Group 12; or CDR1, CDR2, and CDR3 of Group 13 depicted in Table 1A and/or Table IB.

[0004] In some embodiments, the present disclosure provides an antibody or antigen binding fragment thereof that binds (e.g., selectively binds) to CLL-1 at, e.g., an epitope consisting of or comprising the last 50, 40, or 30 C-terminal amino acids of SEQ ID NO: 28. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds (e.g., selectively binds) to CLL-1 at, e.g., an epitope consisting of or comprising amino acids 243 to 275 of SEQ ID NO: 28. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds (e.g., selectively binds) to CLL-1 at, e.g., an epitope consisting of or comprising amino acids 248 to 262 of SEQ ID NO: 28. In some embodiments, the present disclosure provides an antibody or antigen binding fragment thereof that binds (e.g., selectively binds) to CLL-1 at, e.g., an epitope consisting of or comprising amino acids 251 to 260 of SEQ ID NO: 28. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds (e.g., selectively binds) to CLL-1 and does not compete for binding with SC02-357 antibody (e.g., consisting or comprising SEQ ID NO:26). In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds (e.g., selectively binds) to CLL-1 and competes for binding to CLL-1 with an antibody or antigen binding fragment described herein (e.g., comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:

3 to SEQ ID NO: 25). [0005] In some embodiments, the present disclosure provides a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof described herein. In some embodiments, the present disclosure provides a vector comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof described herein. In some embodiments, the present disclosure provides a host cell comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof described herein. In some embodiments, the present disclosure provides a host cell comprising a vector comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof described herein. In some embodiments, the present disclosure provides a method of producing an antibody, or antigen-binding fragment thereof, comprising culturing a host cell, e.g., a host cell comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof described herein, under conditions suitable for expression of an antibody or antigen-binding fragment thereof. In some embodiments, the present disclosure provides a method of treating a CLL-1 associated disease or disorder, the method comprising administering to a subject in need thereof an effective amount of an antibody, or antigen-binding fragment thereof, described herein, e.g., administering a composition (e.g., a pharmaceutical composition) comprising an effective amount of an antibody, or antigen-binding fragment thereof, described herein.

[0006] Other features, objects, and advantages of the present disclosure are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

DEFINITIONS

[0007] A or Am The articles“a” and“an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

[0008] Affinity ·. As used herein, the term“affinity” refers to the characteristics of a binding interaction between an antigen binding moiety (e.g., a single domain antibody described herein) and an antigen target (e.g., CLL-1) and that indicates the strength of the binding interaction. In some embodiments, the measure of affinity is expressed as a dissociation constant (KD). In some embodiments, an antigen binding moiety has a high affinity for an antigen target (e.g., a KD of less than about 10 ⁷ M, less than about 10 ⁸ M, or less than about 10 ⁹ M).

[0009] Approximately or about: As used herein, the term“approximately” or“about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term“approximately” or“about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0010] Antibody. As used herein, the term“antibody” refers to a polypeptide that includes at least one immunoglobulin variable region, e.g., an amino acid sequence that provides an immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term“antibody” encompasses antigen-binding domains or fragments of antibodies (e.g., single domain antibodies, single chain antibodies, Fab, F(ab')2, Fd, Fv, dAb fragments) as well as complete antibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). The light chains of the immunoglobulin can be of types kappa or lambda.

[0011] Antigen target : As used herein, an“antigen target” is any molecule specifically bound by an antigen binding moiety of an antibody described herein. In some embodiments, an antigen target is CLL-1.

[0012] Constant region : As used herein, the term“constant region” refers to a polypeptide that corresponds to, or is derived from, one or more constant region immunoglobulin domains of an antibody. A constant region can include any or all of the following

immunoglobulin domains: a CHI domain, a hinge region, a CH2 domain, a CH3 domain (derived from an IgA, IgD, IgG, IgE, or IgM), and a CH4 domain (derived from an IgE or IgM). [0013] Fc region. As used herein, the term“Fc region” refers to a dimer of two“Fc polypeptides”, each“Fc polypeptide” comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. In some embodiments, an“Fc region” includes two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. “Fc polypeptide” refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also include part or all of the flexible hinge N-terminal to these domains. For IgG,“Fc polypeptide” comprises immunoglobulin domains Cgamma2 (Cy2) and Cgamma3 (Oy3) and the lower part of the hinge between Cgammal (Oyl) and Oy2. Although the boundaries of the Fc polypeptide may vary, the human IgG heavy chain Fc polypeptide is usually defined to comprise residues starting at T223 or C226 or P230, to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, VA). For IgA, Fc polypeptide comprises immunoglobulin domains Calpha2 (Ca2) and Calpha3 (Ca3) and the lower part of the hinge between Calphal (Cal) and Ca2. An Fc region can be synthetic, recombinant, or generated from natural sources such as IVIG.

[0014] Identity·. As used herein, the term“identity” refers to the overall relatedness between between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptides. In some embodiments, nucleic acids or polypeptides are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%,

45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0015] Immunoglobulin single variable domain : The term“immunoglobulin single variable domain” or“single variable domain”, as used herein, means an immunoglobulin variable domain which is capable of specifically binding to an epitope of the antigen without pairing with an additional variable immunoglobulin domain. One example of an

immunoglobulin single variable domain in the meaning of the present disclosure is“domain antibody”, such as the immunoglobulin single variable domains VH and VL (VH domains and VL domains). Another example of an immunoglobulin single variable domain is“VHH domain” (or simply“VHH”) from camelids, as described herein.

[0016] Immunoglobulin variable domain : The term“immunoglobulin variable domain” or“variable domain”, as used herein, means an immunoglobulin domain that is or includes four “framework regions” (referred to in the art and herein as“framework region 1” or“FR1”; as “framework region 2” or“FR2”; as“framework region 3” or“FR3”; and as“framework region 4” or“FR4”, respectively); which framework regions are interrupted by three“complementarity determining regions” or“CDRs” (referred to in the art and herein as“complementarity determining region 1” or“CDR1”; as“complementarity determining region 2” or“CDR2”; and as“complementarity determining region 3” or“CDR3”, respectively). In some embodiments, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0017] Ka. As used herein,“K _a ” refers to an association rate of a particular antigen binding moiety and an antigen target to form an antigen binding moiety/antigen target complex.

[0018] Kd As used herein,“Kd” refers to a dissociation rate of a particular antigen binding moiety/antigen target complex.

[0019] KD: AS used herein,“KD” refers to a dissociation constant, which is obtained from the ratio of Kd to K _a (i.e., Kd/K _a ) and is expressed as a molar concentration (M). KD values can be determined using methods well established in the art, e.g., by using surface plasmon resonance, or using a biosensor system such as a Biacore® system.

[0020] Selective binding : As used herein,“selective binding”,“selectively binds”

“specific binding”, or“specifically binds” refers, with respect to an antigen binding moiety and an antigen target, preferential association of an antigen binding moiety to an antigen target and not to an entity that is not the antigen target. A certain degree of non-specific binding may occur between an antigen binding moiety and a non-target. In some embodiments, an antigen binding moiety selectively binds an antigen target if binding between the antigen binding moiety and the antigen target is greater than 2-fold, greater than 5-fold, greater than 10-fold, or greater than 100- fold as compared with binding of the antigen binding moiety and a non-target. In some embodiments, an antigen binding moiety selectively binds an antigen target if the binding affinity is less than about 10 ⁵ M, less than about 10 ⁶ M, less than about 10 ⁷ M, less than about 10 ⁸ M, or less than about 10 ⁹ M.

[0021] Subject: The term“subject”, as used herein, means any subject for whom diagnosis, prognosis, or therapy is desired. For example, a subject can be a mammal, e.g. , a human or non-human primate (such as an ape, monkey, orangutan, or chimpanzee), a dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle, or cow.

[0022] Therapeutically effective amount: As used herein, the term“therapeutically effective amount” refers to an amount of an antibody or composition described herein that confers a therapeutic effect on a treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). In particular, the “therapeutically effective amount” refers to an amount of an antibody or composition effective to treat, ameliorate, or prevent a particular disease or condition, or to exhibit a detectable therapeutic or preventative effect, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease. A therapeutically effective amount can be administered in a dosing regimen that may comprise multiple unit doses. For any particular antibody or composition, a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and/or unit dose) for any particular subject may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific therapeutic molecule employed; the duration of the treatment; and like factors as is well known in the medical arts.

[0023] Treatment. As used herein, the term“treatment” (also“treat” or“treating”) refers to any administration of an antibody or composition described herein that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.

BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 shows binding of certain VHH clones to CLEC12A.

[0025] Figures 2A-2C show U937 cell binding by certain VHH clones. [0026] Figures 3A-3C show determination of EC50s of certain VHH clones.

[0027] Figures 4A and 4B show E1937 cell binding by certain VHH clones.

[0028] Figures 5A-5D show E1937 cell binding by certain VHH clones.

[0029] Figures 6A-6C show E1937 cell binding by certain VHH clones.

[0030] Figures 7A-7D shows determination of distinct or similar epitopes among certain

VHH clones.

[0031] Figure 8 demonstrates VHH clone 1H1 binds a linear epitope of Clecl2A.

[0032] Figure 9 demonstrates epitope differences between an anti-Clecl2A scFv and certain VHH clones.

[0033] Figures 10A and 10B demonstrate the ability of VHH clone 2H3 to recognize recombinant Clecl2A proteins.

[0034] Figures 11 A and 1 IB demonstrate mass spectrometry identified epitopes of an anti-Clecl2A scFv and VHH clone 2H3 (Note: amino acid numbering in figure based on only extracellular domain amino acids (i.e., the 200 amino acids 65-25 of the full length sequence).

[0035] Figure 12 shows VHH clone 2H3 binds specifically to Clecl2A.

DETAILED DESCRIPTION OF INVENTION

[0036] The present disclosure is based, in part, on the discovery of single domain antibodies that selectively bind to CLL-1/CLEC12A. The disclosure also relates to nucleic acids encoding said antibodies, or antigen-binding fragments thereof, cells comprising such nucleic acids, and methods of use.

Single Domain Antibodies

[0037] Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art known, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. According to one aspect of the disclosure, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in, e.g., WO 94/04678. Such variable domains derived from a heavy chain antibody naturally devoid of light chain is referred to herein as a“VHH” or“nanobody”. Such a VHH can be derived from antibodies raised in Camelidae species, for example in camel, dromedary, llama, vicuna, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the disclosure.

[0038] The amino acid residues of VHH domains from Camelids are numbered according to the general numbering for VH domains given by Kabat et ah,“Sequence of proteins of immunological interest”, US Public Health Services, NIH (Bethesda, MD), Publication No 91-3242 (1991); see also Riechmann et ah, J. Immunol. Methods 231 :25-38 (1999). According to this numbering, FR1 comprises the amino acid residues at positions 1-30, CDR1 comprises the amino acid residues at positions 31-35, FR2 comprises the amino acids at positions 36-49, CDR2 comprises the amino acid residues at positions 50-65, FR3 comprises the amino acid residues at positions 66-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113.

[0039] It should be noted, however (as is well known in the art for VH domains and for

VHH domains), that the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. [0040] Alternative methods for numbering the amino acid residues of VH domains, which methods can also be applied in an analogous manner to VHH domains, are known in the art. However, in the present disclosure, claims and figures, the numbering according to Kabat and applied to VHH domains as described above will be followed, unless indicated otherwise.

[0041] In some embodiments, the disclosure provides a CLL-1 binding antibody that is or includes a VHH having the amino acid sequence of any one of SEQ ID Nos:3-25, or a fragment thereof (e.g., a CLL-1 binding fragment thereof). As indicated in the listing of sequences provided herein, each of SEQ ID Nos:3-25 includes VHH amino acids at the N- terminus, and the following amino acids at the C-terminus: (i) a linker of 9 amino acids

(TSGPGGQGA), (ii) a myc-tag (EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS). In some

embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH having a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks one or more of (i)-(v) (and/or lacks a portion of one or more of (i)-(v)). In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH having a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks one or more of the C-terminal amino acids

T SGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25. In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL- 1, that is or includes a VHH having a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks all of the C-terminal amino acids T S GPGGQGAEQKLI SEEDLGAHHHHHHGA S depicted in each of SEQ ID Nos:3-25.

[0042] In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH having a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks one or more of (i)-(v) (and/or lacks a portion of one or more of (i)-(v)), and wherein the portion lacks one or more (e.g., 1, 2, 3, 4, 5, or more), additional amino acids (i.e., other than an amino acid included in (i)-(v)). In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH having a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks one or more of the C-terminal amino acids

T SGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25, and wherein the portion lacks one or more (e.g., 1, 2, 3, 4, 5, or more), additional amino acids. In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL- 1, that is or includes a VHH having a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks all of the C-terminal amino acids T S GPGGQGAEQKLI SEEDLGAHHHHHHGA S depicted in each of SEQ ID Nos:3-25, and wherein the portion lacks one or more (e.g., 1, 2, 3, 4, 5, or more), additional amino acids.

[0043] In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3- 25, wherein the portion lacks one or more of (i)-(v) (and/or lacks a portion of one or more of (i)- (v)). In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH having an amino acid sequence that is at least 85%,

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks one or more of the C-terminal amino acids

T SGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25. In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL- 1, that is or includes a VHH having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks all of the C-terminal amino acids T SGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25.

[0044] In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3- 25, wherein the portion lacks one or more of (i)-(v) (and/or lacks a portion of one or more of (i)- (v)), and wherein the portion lacks one or more (e.g., 1, 2, 3, 4, 5, or more), additional amino acids (i.e., other than an amino acid included in (i)-(v)). In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks one or more of the C-terminal amino acids T S GPGGQGAEQKLI SEEDLGAHHHHHHGA S depicted in each of SEQ ID Nos:3-25, and wherein the portion lacks one or more (e.g., 1, 2, 3, 4, 5, or more), additional amino acids.

In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion (e.g., a CLL-1 binding portion) of the amino acid sequence of any one of SEQ ID Nos:3-25, wherein the portion lacks all of the C-terminal amino acids TSGPGGQGAEQKLISEEDLGAHHHHHHGAS depicted in each of SEQ ID Nos:3-25, and wherein the portion lacks one or more (e.g., 1, 2, 3, 4, 5, or more), additional amino acids.

[0045] In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH comprising at least one CDR (e.g., CDR1, CDR2, and/or CDR3) depicted in any one of SEQ ID Nos:3-25. In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH comprising a portion of at least one CDR (e.g., CDR1, CDR2, and/or CDR3) depicted in any one of SEQ ID Nos:3-25, wherein the portion lacks 1, 2, 3, 4, 5, or more amino acids of a CDR depicted in any one of SEQ ID Nos:3-25. In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH comprising at least one CDR that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a CDR (e.g., CDR1, CDR2, and/or CDR3) depicted in any one of SEQ ID Nos:3-25. In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of at least one CDR (e.g., CDR1, CDR2, and/or CDR3) depicted in any one of SEQ ID Nos:3-25, wherein the portion lacks 1, 2, 3, 4, 5, or more amino acids of a CDR depicted in any one of SEQ ID Nos: 3 -25.

[0046] In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH comprising CDR1, CDR2, and/or CDR3 of any one of Groups 1-13 depicted in Table 1A and/or Table IB. In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH comprising (i) CDR1 and CDR2; (ii) CDR2 and CDR3; (iii) CDR1 and CDR3; or (iv) CDR1, CDR2, and CDR3 of any one of Groups 1-13 depicted in Table 1A and/or Table IB (e.g., wherein the CDRs are from one particular Group, or wherein the CDRs are selected from two or more different Groups). In some embodiments, the disclosure provides an antibody that binds (e.g., selectively binds) CLL-1, that is or includes a VHH comprising CDR1, CDR2, and CDR3 of Group 1; CDR1, CDR2, and CDR3 of Group 2; CDR1, CDR2, and CDR3 of Group 3; CDR1, CDR2, and CDR3 of Group 4; CDR1, CDR2, and CDR3 of Group 5; CDR1, CDR2, and CDR3 of Group 6; CDR1, CDR2, and CDR3 of Group 7; CDR1, CDR2, and CDR3 of Group 8; CDR1, CDR2, and CDR3 of Group 9; CDR1, CDR2, and CDR3 of Group 10; CDR1, CDR2, and CDR3 of Group 11; CDR1, CDR2, and CDR3 of Group 12; or CDR1, CDR2, and CDR3 of Group 13, as depicted in Table 1 A and/or Table IB.

Table 1A:

Table IB: CDRs as identified based on IMGT numbering and ANARCI software

(http://opig.stats.ox.ac.uk/webapps/sabdab-sabpred/ANARCI.ph p)

[0047] In some embodiments, the present disclosure provides an antibody or antigen binding fragment thereof that binds (e.g., selectively binds) to CLL-1 at, e.g., an epitope in the last 50, 40, or 30 amino acids of SEQ ID NO: 28. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds (e.g., selectively binds) to CLL-1 at, e.g., an epitope within amino acids 243 to 275 of SEQ ID NO: 28. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds (e.g., selectively binds) to CLL-1 at, e.g., an epitope within amino acids 248 to 262 of SEQ ID NO: 28. In some embodiments, the present disclosure provides an antibody or antigen binding fragment thereof that binds (e.g., selectively binds) to CLL-1 at, e.g., an epitope within amino acids 251 to 260 of SEQ ID NO: 28. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds (e.g., selectively binds) to CLL-1 and does not compete for binding with SC02-357. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds (e.g., selectively binds) to CLL-1 and that competes for binding to CLL-1 with an antibody or antigen binding fragment described herein (e.g., comprising a sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 25).

[0048] As will be understood by those of skill in the art, any such CDR sequence may be readily combined, e.g., using molecular biology techniques, with any other polypeptide (e.g., antibody) sequences or domains provided herein or otherwise known in the art, including any framework regions, CDRs, or constant domains, or portions thereof as disclosed herein or otherwise known in the art, as may be present in an antibody or binding molecule of any format as disclosed herein or otherwise known in the art.

[0049] Antibodies or fragments can be produced by any method known in the art for synthesizing antibodies (see, e.g., Harlow et ak, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Brinkman et ak, 1995, J. Immunol. Methods 182:41-50; WO 92/22324; WO 98/46645). Chimeric antibodies can be produced using methods described in, e.g., Morrison, 1985, Science 229:1202, and humanized antibodies by methods described in, e.g., U.S. Pat. No. 6,180,370.

[0050] Additional antibodies of compositions and methods described herein are bispecific antibodies and multivalent antibodies, as described in, e.g., Segal et ak, J. Immunol. Methods 248: 1-6 (2001); and Tutt et ak, J. Immunol. 147: 60 (1991).

Fusion Proteins and Conjugates

[0051] In some embodiments, the disclosure provides fusion proteins comprising (i) one or more single domain antibodies, or antigen-binding fragments thereof, described herein (e.g., one or more CDRs described herein), and (ii) one or more additional polypeptides. For example, a fusion protein can include one or more single domain antibodies described herein and a constant region or Fc region described herein. In some embodiments, one or more single domain antibodies, or antigen-binding fragments thereof, described herein (e.g., one or more CDRs described herein) can be conjugated noncovalently or covalently, e.g., fused, to an antigen (e.g., an antigen target for a cellular therapeutic, e.g., a CAR-T cell or antibody drug conjugate) as described in, e.g., WO2017/075537, WO2017/075533, WO2018156802, and WO2018156791.

[0052] In some embodiments, the disclosure provides a fusion protein comprising one or more VHH as described herein and one or more additional polypeptides. In some embodiments, an additional polypeptide comprises an additional antibody or fragment thereof. Additional antibodies include, e.g., intact IgG, IgE and IgM, bi- or multi- specific antibodies (e.g.,

Zybodies®, etc), single chain Fvs, polypeptide-Fc fusions, Fabs, cameloid antibodies, masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuti cals (“SMIPsTM”), single chain or Tandem diabodies (TandAb®), VHHs (including but not limited to those described in the present disclosure), Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, a DART, a TCR-like antibody, Adnectins®, Affilins®, Trans bodies®, Affibodies®, a TrimerX®, MicroProteins, Fynomers®, Centyrins®, and a

KALBITOR®. Exemplary additional antibodies are listed in Table 4. In some embodiments, an additional antibody targets PD-1, TIM-3, LAG-3, IDO, A2AR, TGFbeta, CD47, or another protein involved in an immunosuppressive pathway.

[0053] Table 4:

[0054] In some embodiments, an additional polypeptide comprises or consists of all or a portion of a tumor associated antigen (TAA) or tumor specific antigen (TSA). Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-l/MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other tumor antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, erbB, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1,

NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha- fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, MUC16, IL13Ra2, FRa, VEGFR2, Lewis Y, FAP, EphA2, CEACAM5, EGFR, CA6, CA9, GPNMB, EGP1, FOLR1, endothelial receptor, STEAPl, SLC44A4, Nectin-4, AGS- 16, guanalyl cyclase C, MUC-1, CFC1B, integrin alpha 3 chain (of a3bl, a laminin receptor chain), and TPS.

[0055] In some embodiments, an additional polypeptide comprises or consists of all or a portion of a tumor antigen selected from CD 19, CD20, CD22, CD30, CD72, CD 180, CD171 (LI CAM), CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166,

CD71, CLL-1/CLECK12A, ROR1, Glypican 3 (GPC3), Mesothelin, CD33/IL3Ra, c-Met,

PSCA, PSMA, Glycolipid F77, EGFRvIII, GD-2, MY-ESO-1, and MAGE A3. [0056] In some embdoiments, an additional polypeptide comprises or consists of all or a portion of a B cell specific marker selected from CD 19, CD20, CD21, CD22, CD23, CD24, CD40, CD72, CD 180, ROR1, BCMA, CD79a, and CD79b (see, e.g., LeBien et al., Blood 112: 1570-1580 (2008)).

[0057] In some embodiments, the disclosure provides antibodies conjugated to a therapeutic moiety, such as a cytotoxin, a biologically active protein (e.g., one or more peptide or one or more cytokine) or a radioisotoperadiotoxin. Such conjugates are referred to herein as “immunococonjugates”. Immunoconjugates that include one or more cytotoxins are referred to as“immunotoxins”. A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

[0058] Therapeutic agents that can be conjugated also include, for example,

antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[0059] Other preferred examples of therapeutic cytotoxins that can be conjugated to antibodies described herein include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof.

[0060] Cytotoxins can be conjugated to antibodies using linker technology available in the art. Examples of linker types that have been used to conjugate a cytotoxin to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers. A linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

[0061] For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see also Saito, G. etal. (2003) Adv. Drug Deliv. Rev. 55: 199- 215; Trail, P. A. et al. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I. and

Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3: 1089-1091; Senter, P. D. and Springer, C J. (2001) Adv. Drug Deliv. Rev. 53:247-264.

[0062] Antibodies described herein can also be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for use diagnostically or

therapeutically include, but are not limited to, iodine ¹³¹ , indium ¹¹¹ , yttrium ⁹⁰ and lutetium ¹⁷⁷ . Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™ (Spectrum

Pharmaceuticals) and Bexxar™ (formally sold by GlaxoSmithKline), and similar methods can be used to prepare radioimmunoconjugates using antibodies described herein.

Nucleotide Sequences

[0063] The present disclosure includes nucleotide sequences encoding one or more antibodies described herein (e.g., a VHH described herein), or portion thereof (e.g., one or more CDRs described herein), and/or one or more fusion proteins described herein. In various instances, such nucleotide sequences may be present in a vector. In various instances such nucleotides may be present in the genome of a cell, e.g., a cell of a subject in need of treatment or a cell for production of an antibody, e.g. a mammalian cell for production of an antibody.

CLL-1

[0064] Human C-type lectin-like molecule-1 (CLL-1), also known as MICL or

CLEC12A, is a type II transmembrane glycoprotein and member of the large family of C-type lectin-like receptors involved in immune regulation. CLL-1 has previously been identified from myeloid-derived cells. The intracellular domain of CLL-1 contains an immunotyrosine-based inhibition motif (ITIM) and a YXXM motif. Phosphorylation of ITIM-containing receptors on a variety of cells results in inhibition of activation pathways through recruitment of protein tyrosine phosphatases SHP-1, SHP-2 and SHIP. The YXXM motif has a potential SH2 domain binding site for the p85 subunit of PI-3 kinase, 13 which has been implicated in cellular activation pathways, revealing a potential dual role of CLL-1 as an inhibitory and activating molecule on myeloid cells. Indeed, association of CLL-1 with SHP-1 and SHP-2 has been demonstrated experimentally in transfected and myeloid-derived cell lines.

[0065] The pattern of expression of CLL-1 in hematopoietic cells is restricted. It is found in particular in myeloid cells derived from peripheral blood and bone marrow, as well as in the majority of AML blasts. A recent study indicated that CLL-1 is also present on the majority of leukemic stem cells in the CD34+/CD38- compartment in AML but absent from CD34+/CD38- cells in normal and in regenerating bone marrow controls, which aids the discrimination between normal and leukemic stem cells. (See, e.g., Zhao et ah, Haematologica 95:71-78 (2010); Bakker et al., Cancer Res. 64:8443-8450 (2004)).

[0066] The nucleotide and protein sequences of CLL-1 are known for many species. For example, the human sequences can be found at Genbank accession number AF247788.1 (coding sequence shown in SEQ ID NO:l) and Uniprot accession number Q5QGZ9 (SEQ ID NO:2). For the human CLL-1 protein shown as SEQ ID NO:2, the extracellular domain comprises approximately amino acids 65-265, the transmembrane domain comprises approximately amino acids 44-64, and the cytoplasmic domain comprises approximately amino acids 1-43. The stalk domain of human CLL-1 spans amino acids 65-139, and the C lectin domain spans amino acids 140-249, both with reference to the sequence shown in SEQ ID NO:2. One of ordinary skill will understand that CLL-1 variants (e.g., species homologs, allelic variants, etc.) can be optimally aligned, e.g., for identification of conserved residues and domains. CLL-1 Associated Disorders

[0067] The antibodies and/or fusion proteins of the disclosure can be used, e.g., to detect and/or treat CLL-1 associated disorders, i.e., diseases correlated with elevated or reduced cell surface expression of CLL-1 as compared to CLL-1 expression in a standard control (e.g., a normal, non-disease, non-cancer cell). CLL-1 expression is normally limited to myeloid lineage cells, e.g., dendritic cells, granulocytes, and monocytes in the peripheral blood and spleen. In some instances, elevated CLL-1 levels are associated with cancer, in particular, in hematopoietic CSCs (e.g., LSCs), and in myeloproliferative disorders, including leukemias such as AML (acute myelogenous or myeloproliferative leukemia), MDS (myelodysplastic syndrome), myelofibrosis, CMML (chronic myelomonocytic leukemia), multiple myeloma, plasmacytoma, and CML (chronic myelogenous or myeloproliferative leukemia). See, e.g., Morsink et al., Blood Rev. 2018 Nov 1. pii: S0268-960X(18)30072-9. doi: 10.1016/j blre.2018.10.003. [Epub]; Bakker et al., Cancer Res. 64:8443-8450 (2004); Van Rhenen et al., Blood 110:2659-66 (2007); Zhao et al., Haematologica 95:71-78 (2010); Van Rhenen et al., Leukemia 21 : 1700-7 (2007); and Herrmann et al., Haematologica 97:219-26 (2012).

[0068] AML cells can be characterized and distinguished from other cells by detecting cell surface marker expression. Aside from being CLL-1+, AML cells can be CD33+(though some are CD33-), CD45+, and CDw52+. AML blasts (including LSCs) are typically

CD34+CD38-. HSCs and LSCs can be characterized by expression of CD34, but the former do not express CLL-1. MDS cells can be characterized by expression of CDS, CD7, CD13, and CD34. CML cells can be characterized by expression of 7-ADD, CD33, CD34, and CD38.

[0069] Myelodysplastic Syndromes (MDS) include a group of closely-related blood formation disorders, in which the bone marrow shows qualitative and quantitative changes suggestive of a preleukemic process, but having a chronic course that does not necessarily terminate as acute leukemia. A variety of terms, including preleukemia, refractory anemia, refractory dysmyelopoietic anemia, smoldering or subacute leukemia, dysmyelopoietic syndrome (DMPS), and myelodysplasia, have all been used to describe MDS. These conditions can be characterized by a cellular marrow with impaired maturation (dysmyelopoiesis) and a reduction in the number of blood cells. DMPS can be characterized by presence of megablastoids, megarkaryocyte dysplasia, and an increase in number of abnormal blast cells, reflective of enhanced granulocyte maturation process. Patients with DMPS typically show chromosomal abonormalities similar to those found in acute myeloid leukemia and progress to acute myeloid leukemia in a certain fraction of afflicted patients.

[0070] Chronic myeloproliferative disorders are a collection of conditions that can be characterized by increased number of mature and immature granulocytes, erythrocytes, and platelets. Chronic myeloproliferative disorders can transition to other forms within this group, with a tendency to terminate in acute myeloid leukemia. Specific diseases within this group include polycythemia vera, chronic myeloid leukemia, agnogenic myeloid leukemia, essential thrombocythemia, and chronic neutrophilic leukemia.

[0071] Myelofibrosis can be characterized by scarring of the bone marrow that can result in reduced number of red and white blood cells, and platelets. Myelofibrotic scarring can result from leukemia, but can have other causes, such as thrombocytosis or adverse drug effects.

[0072] In various instances, an antibody and/or fusion protein described herein treats, alleviates, reduces the prevalence of, reduces the frequency of, or reduces the level or amount of one or more symptoms or biomarkers of a CLL-1 -associated disorder. Specific symptoms and progression of symptoms vary among subjects. Thus, in some embodiments, an antibody and/or fusion protein described herein is administered to a subject in need thereof, e.g., a subject having a CLL-1 -associated disorder.

[0073] In various instances, administration of an antibody and/or fusion protein described herein results in a decrease in the prevalence, frequency, level, and/or amount of one or more symptoms or biomarkers of a CLL-1 -associated disorder, e.g., a decrease of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of one or more symptoms or biomarkers as compared to a prior

measurement in the subject or to a reference value.

[0074] In some embodiments, an effective dose of an antibody and/or fusion protein as described herein may be, e.g., less than 1,000 mg/dose, e.g., less than 900 mg/dose, 800 mg/dose, 700 mg/dose, 600 mg/dose, 500 mg/dose, 550 mg/dose, 400 mg/dose, 350 mg/dose, 300 mg/dose, 200 mg/dose, 100 mg/dose, 50 mg/dose, 25 mg/dose, or less. Alternatively or in combination with a dosage as disclosed herein, an antibody and/or fusion protein as described herein may be effectively or usefully administered at a frequency that is less than once per week, e.g., less than once every week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or year.

[0075] In some embodiments, an antibody and/or fusion protein described herein can be used in a number of diagnostic and/or therapeutic applications. For example, detectably-labeled versions of antibodies as described herein can be used in assays to detect the presence or amount of CLL-1 in a sample (e.g., a biological sample). Antibodies and/or fusion proteins described herein can be used in in vitro assays for studying inhibition of CLL-1 activity. In some embodiments, an antibody and/or fusion protein described herein can be used as a positive control in an assay designed to identify additional novel compounds that inhibit CLL-1 or otherwise are useful for treating a CLL-1 -associated disorder.

[0076] Antibodies and/or fusion proteins described herein may be used in monitoring a subject, e.g., a subject having, suspected of having, at risk of developing, or under treatment for one or more CLL-1 -associated disorders. Monitoring may include determining the amount or activity of CLL-1 in a subject, e.g., in the serum of a subject. In some embodiments, the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration of an antibody and/or fusion protein as described herein. The subject can be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation can include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g., adding or dropping any of the treatments for a CLL-1 -associated disorder described herein. Measuring Interactions of Antibodies and CLL-1

[0077] The binding properties of an antibody described herein to CLL-1 can be measured by methods known in the art, e.g., one of the following methods: BIACORE analysis, Enzyme Linked Immunosorbent Assay (ELISA), x-ray crystallography, sequence analysis and scanning mutagenesis. The binding interaction of an antibody and CLL-1 can be analyzed using surface plasmon resonance (SPR). SPR or Biomolecular Interaction Analysis (BIA) detects bio-specific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface. The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S. Pat. No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338- 2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line resources provide by BIAcore International AB (Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.) can also be used.

[0078] Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (KD), and kinetic parameters, including K _0n and Koff, for the binding of an antibody to CLL-1. Such data can be used to compare different molecules. Information from SPR can also be used to develop structure-activity relationships (SAR). Variant amino acids at given positions can be identified that correlate with particular binding parameters, e.g., high affinity.

[0079] In certain embodiments, an antibody described herein exhibits high affinity for binding CLL-1. In various embodiments, KD of an antibody as described herein for CLL-1 is less than about 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , lO ¹⁰ , lO ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , or 10 ¹⁵ M. In certain instances, KD of an antibody as described herein for CLL-1 is between 0.001 and 1 nM, e.g., 0.001 nM, 0.005 nM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM.

Formulations and Administration [0080] The present disclosure includes compositions, e.g., pharmaceutical compositions, containing one or more antibodies and/or fusion proteins described herein, formulated together with a pharmaceutically acceptable carrier. Methods well known in the art for making formulations are found, for example, in“Remington: The Science and Practice of Pharmacy” (20th ed., ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.). As used herein,“pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. For example, the carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the antibody may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0081] A pharmaceutical composition may include a pharmaceutically acceptable anti oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium

metabi sulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid,

ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

[0082] Prevention of presence of microorganisms can be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0083] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is

contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0084] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0085] Sterile injectable solutions can be prepared by incorporating an antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0086] The amount of antibody and/or fusion protein described herein that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of antibody and/or fusion protein that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of antibody, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of antibody in combination with a pharmaceutically acceptable carrier. [0087] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of

antibody calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the antibody and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an antibody for the treatment of sensitivity in individuals.

[0088] For administration of an antibody and/or fusion protein disclosed herein, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the subject body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight or 20 mg/kg body weight or within the range of 1-20 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months, or with a short administration interval at the beginning (such as once per week to once every three weeks), and then an extended interval later (such as once a month to once every three to 6 months).

[0089] Alternatively, antibody and/or fusion protein can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody and/or fusion protein in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0090] Actual dosage levels of antibody and/or fusion protein in the pharmaceutical compositions of the disclosure may be varied so as to obtain an amount of antibody and/or fusion protein that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0091] A composition of the disclosure can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0092] Alternatively, a composition can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. [0093] The antibody and/or fusion protein can be prepared with carriers that will protect against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,

polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0094] Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the disclosure can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. No. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present disclosure include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for

administering medicaments through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0095] In some embodiments, an antibody and/or fusion protein described can be admininstered to a subject using a vector, e.g., a viral vector, e.g., using known methods. In some embodiments, a viral vector can be used to introduce an antibody and/or fusion protein into a cancer cell (e.g., a tumor cell). Introduction of such antibody and/or fusion protein can increase susceptibility to a subject’s immune system and/or one or more additional therapeutic agents (see, e.g., WO2017/075533).

[0096] A nucleic acid sequence encoding an antibody and/or fusion protein described herein can be cloned into a number of types of vectors. For example, a nucleic acid can be cloned into a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Other vectors can include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and viral vectors. In other examples, the vector can be a foamy viral (FV) vector, a type of retroviral vector made from spumavirus. Viral vector design and technology is well known in the art as described in Sambrook et al, (Molecular Cloning: A Laboratory Manual, 2001), and in other virology and molecular biology manuals.

[0097] A number of viral based systems have been developed for gene transfer into mammalian cells. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, poxviruses, herpes simplex 1 virus, herpes virus, oncoviruses (e.g., murine leukemia viruses), and the like. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193). Lentiviral and Retroviral transduction can be enhanced by the addition of polybrene (SantaCruz sc-134220; Millipore TR- 1003-G; Sigma 107689), a cationic polymer (also known as hexamehtrine bromide) that is used to increase the efficiency of the retrovirus transduction.

[0098] Retroviruses are enveloped viruses that belong to the viral family Retroviridae.

Once in a host’s cell, the virus replicates by using a viral reverse transcriptase enzyme to transcribe its RNA into DNA. The retroviral DNA replicates as part of the host genome, and is referred to as a provirus. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject in vivo. A number of retroviral systems are known in the art, (see, e.g., U.S. Pat Nos. 5,994,136, 6,165, 782, and 6,428,953).

[0099] Retroviruses include the genus of Alpharetrovirus (e.g., avian leukosis virus), the genus of Betaretrovirus; (e.g., mouse mammary tumor virus) the genus of Deltaretrovirus (e.g., bovine leukemia virus and human T-lymphotropic virus), the genus of Epsilonretrovirus (e.g., Walleye dermal sarcoma virus), and the genus of Lentivirus. In some embodiments, a retrovirus is a lentivirus a genus of viruses of the Retroviridae family, e.g., characterized by a long incubation period. Lentiviruses are unique among the retroviruses in being able to infect non- dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so can be used as an efficient gene delivery vector. In some examples, a lentivirus can be, but not limited to, human immunodeficiency viruses (HIV-1 and HIV-2), simian

immunodeficiency virus (S1V), feline immunodeficiency virus (FIV), equine infections anemia (EIA), and visna virus. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0100] In some embodiments, a vector is an adenovirus vector. Adenoviruses are a large family of viruses containing double stranded DNA. They replicate the DNA of the host cell, while using a host’s cell machinery to synthesize viral RNA DNA and proteins. Adenoviruses are known in the art to affect both replicating and non-replicating cells, to accommodate large transgenes, and to code for proteins without integrating into the host cell genome.

[0101] In some embodiments, an AAVP vector is used. An AAVP vector is a hybrid of prokaryotic-eukaryotic vectors, which are chimeras of genetic cis-elements of recombinant adeno-associated virus and phage. An AAVP combines selected elements of both phage and AAV vector systems, providing a vector that is simple to produce in bacteria and can exhibit little or no packaging limit, while allowing infection of mammalian cells combined with integration into the host chromosome. Vectors containing many of the appropriate elements are commercially available, and can be further modified by standard methodologies to include the necessary sequences. Among other things, AAVPs do not require helper viruses or trans-acting factors. In addition, the native tropism of AAV for mammalian cells is eliminated since there is not AAV capsid formation. Other methods and details are in U.S. Patent 8,470,528 and Hajitou A. et ak, Cell, 125: 358-398.

[0102] In some embodiments, a human papilloma (HPV) pseudovirus is used. DNA plasmids can be packaged into papillomavirus LI and L2 capsid protein to generate pseudovirion that can efficiently deliver DNA. The encapsulation can protect the DNA from nucleases and provides a targeted delivery with a high level of stability. Many of the safety concerns associated with the use of viral vectors can be mitigated with an HPV pseudovirus. Other methods and examples are in Hung, C., et al.,Plos One, 7:7(e40983); 2012, U.S. Patent

8,394,411, and Kines, R., et al Int J of Cancer, 2015. [0103] In some embodiments, an oncolytic virus is used. Oncolytic virus therapy can selectively replicate the virus in cancer cells, and can subsequently spread within a tumor, e.g., without affecting normal tissue. Alternatively, an oncolytic virus can preferentially infect and kill cells without causing damage to normal tissues. Oncolytic viruses can also effectively induce immune responses to themselves as well as to the infected tumor cell. Typically, oncolytic viruses fall into two classes: (I) viruses that naturally replicate preferentially in cancer cells and are nonpathogenic in humans. Exemplary class (I) oncolytic viruses include autonomous parvoviruses, myxoma virus (poxvirus), Newcastle disease virus (NDV;

paramyxovirus), reovirus, and Seneca valley virus (picornavirus). A second class (II), includes viruses that are genetically manipulated for use as vaccine vectors, including measles virus (paramyxovirus), poliovirus (picornavirus), and vaccinia virus (poxvirus). Additionally, oncolytic viruses may include those genetically engineered with mutations/deletions in genes required for replication in normal but not in cancer cells including adenovirus, herpes simplex virus, and vesicular stomatitis virus. Oncolytic viruses can be used as a viral transduction method due to their low probability of genetic resistance because they can target multiple pathways and replicate in a tumor-selective method. The viral dose within a tumor can increase over time due to in situ viral amplification (as compared to small molecule therapies which decrease with time), and safety features can be built in (i.e., drug and immune sensitivity).

Combination Therapies

[0104] In various embodiments, an antibody and/or fusion protein as described herein may be included in a course of treatment that further includes administration of at least one additional agent to a subject. In various instances, an additional agent administered in combination with an antibody and/or fusion protein as described herein can be cytarabine (cytosine arabinoside, or ara-C) and/or antharcycline drugs such as doxorubicin, daunorubicin, daunomycin, idarubicin and mitoxantrone; other chemotherapeutic drugs such as Hydroxyurea (Hydrea®), Decitabine (Dacogen®), Cladribine (Leustatin®, 2-CdA), Fludarabine (Fludara®), Topotecan, Etoposide (VP- 16), 6-thioguanine (6-TG), corticosteroid drugs, such as prednisone or dexamethasone (Decadron®), methotrexate (MTX), 6-mercaptopurine (6-MP) or Azacitidine (Vidaza®), all-trans-retinoic acid (ATRA), tretinoin, or Vesanoid® and arsenic trioxide (ATO, Trisenox®). In some embodiments, a fusion protein described herein (e.g., a fusion protein consisting of or comprising one or more VHH and one or more tumor antigen) is administered in combination with a cellular therapeutic (e.g., a CAR-T cell) or antibody drug conjugate that binds to such one or more tumor antigen as described in, e.g., WO2017/075537,

WO2017/075533 , WO2018156802, and WO2018156791.

[0105] In various instances, an additional agent administered in combination with an antibody and/or fusion protein as described herein may be administered at the same time as an antibody and/or fusion protein, on the same day as an antibody and/or fusion protein, or in the same week as an antibody and/or fusion protein. In various instances, an additional agent administered in combination with an antibody and/or fusion protein as described herein may be administered in a single formulation with an antibody and/or fusion protein. In certain embodiments, an additional agent administered in a manner temporally separated from administration of an antibody and/or fusion protein as described herein, e.g., one or more hours before or after, one or more days before or after, one or more weeks before or after, or one or more months before or after administration of an antibody and/or fusion protein. In various embodiments, the administration frequency of one or more additional agents may be the same as, similar to, or different from the administration frequency of an antibody and/or fusion protein as described herein.

[0106] When compositions are to be used in combination with a second active agent, the compositions can be co-formulated with the second agent or the compositions can be formulated separately from the second agent formulation. For example, the respective pharmaceutical compositions can be mixed, e.g., just prior to administration, and administered together or can be administered separately, e.g., at the same or different times.

[0107] In some embodiments, combined administration of an antibody and/or fusion protein described herein and an additional agent results in an improvement in a condition or a symptom thereof to an extent that is greater than one produced by either the antibody (or fusion protein) or the additional agent alone. The difference between the combined effect and the effect of each agent alone can be a statistically significant difference. In some embodiments, combined administration of an antibody and/or fusion protein described herein and an additional agent allows administration of the additional agent at a reduced dose, at a reduced number of doses, and/or at a reduced frequency of dosage compared to a dosing regimen for the additional agent, e.g., a standard dosing regimen approved for the additional agent.

Kits

[0108] An antibody, or antigen-binding fragment thereof, and/or fusion described herein

(e.g., a pharmaceutical composition comprising an antibody or antigen-binding fragment and/or fusion protein) can be provided in a kit. In some instances, the kit includes (a) a container that contains an antibody and/or fusion protein described herein (e.g., a pharmaceutical composition comprising an antibody and/or fusion protein described herein) and, optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of an antibody and/or fusion protein, e.g., for therapeutic benefit.

[0109] The informational material of a kit is not limited in its form. In some instances, the informational material can include information about production of an antibody and/or fusion protein, amino acid of an antibody and/or fusion protein, nucleic acid encoding an antibody and/or fusion protein, molecular weight of an antibody and/or fusion protein, concentration, date of expiration, batch or production site information, and so forth. In other situations, the informational material relates to methods of administering an antibody and/or fusion protein, e.g., in a suitable amount, manner, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). The method can be a method of treating a subject having a CLL-1 associated disorder.

[0110] In some cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. The informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In other instances, the informational material of the kit is contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about an antibody therein and/or their use in the methods described herein. The informational material can also be provided in any combination of formats.

[0111] In addition to an antibody and/or fusion protein described herein, a kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. A kit can also include other agents, e.g., a second or third agent, e.g., other therapeutic agents. The components can be provided in any form, e.g., liquid, dried or lyophilized form. The components can be substantially pure (although they can be combined together or delivered separate from one another) and/or sterile. When the components are provided in a liquid solution, the liquid solution can be an aqueous solution, such as a sterile aqueous solution. When the components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent.

The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.

[0112] A kit can include one or more containers for an antibody (and/or fusion protein) and/or other agents. In some cases, a kit contains separate containers, dividers or compartments for an antibody and/or fusion protein and informational material. For example, an antibody and/or fusion protein can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other situations, the separate elements of a kit are contained within a single, undivided container. For example, an antibody and/or fusion protein can be contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some cases, a kit can include a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of an antibody and/or fusion protein. Containers can include a unit dosage, e.g., a unit that includes an antibody and/or fusion protein. For example, a kit can include a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a unit dose. The containers of kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.

[0113] A kit can optionally include a device suitable for administration of an antibody and/or fusion protein, e.g., a syringe or other suitable delivery device. A device can be provided preloaded with an antibody and/or fusion protein, e.g., in a unit dose, or can be empty, but suitable for loading.

Recombinant Gene Technology

[0114] In accordance with the present disclosure, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are described in the literature (see, e.g ., Sambrook, Fritsch & Maniatis,

Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));

Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0115] Recombinant expression of a gene, such as a nucleic acid encoding a polypeptide, such as an antibody described herein, can include construction of an expression vector containing a nucleic acid that encodes the polypeptide. Once a polynucleotide has been obtained, a vector for the production of the polypeptide can be produced by recombinant DNA technology using techniques known in the art. Known methods can be used to construct expression vectors containing polypeptide coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0116] An expression vector can be transferred to a host cell by conventional techniques, and the transfected cells can then be cultured by conventional techniques to produce

polypeptides. A number of viral based systems have been developed for gene transfer into mammalian cells. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, poxviruses, herpes simplex 1 virus, herpes virus, oncoviruses (e.g., murine leukemia viruses, vaccinia virus), and the like. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193). For example, lymphocytes (e.g., T cells) can be transduced using a lentiviral or retroviral vector. An oncolytic viral vector (e.g., adenovirus, vaccinia, AAV) can be used to transduce a tumor cell (e.g., which can secrete an antibody described herein). See, e.g., WO2017/075533.

[0117] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

EXAMPLES

[0118] The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually performed or data were actually obtained.

Example 1 - Assessment of VHH antibodies

[0119] The clones described in the Examples have the amino acid sequences noted in

Table 2.

Table 2:

[0120] Around 184 sdAb samples were screened for binding to CLEC12A (also known as CLL-1), coated onto a plate, by ELISA. Plates were coated with 1 pg/mL human CLEC12A in PBS (O/N 4C), then blocked with 5% milk/PBST (2hr, room temp); added bacterially expressed sdAbs diluted in 1 : 1 in Block (lhr), washed 5X with PBST, detected with mouse anti- myc-Tag monoclonal antibody (mAb) for lhr, followed by goat anti-mouse IgG-HRP (lhr), both in Block, washed 5X with PBST between antibodies, and plate developed 30 minutes. Each plate included 4 controls / 92 samples per plate. About 32% of sdAb screened were ELISA positive. Figure 1 shows binding data for certain clones.

[0121] U937 cell binding by VHH clones was assessed. Briefly, the method included:

U937 cells (2.5c10 ^L 5) were Fc blocked (human Fc block, BD Cat. # BDB564220) for lOmin at room temperature, then washed once with FACS buffer (PBS + 1% BSA + 0.1% Sodium Azide) by spinning at 500G for 2min; VHH clones (3pg/ml as final cone) in FACS buffer were added to the cell pellets and incubated for 30min at 4°C, then washed twice with FACS buffer by spinning at 500G for 2min; anti-His-PE (R&D Systems, Cat. # IC050P) was added to cells; the mixture was incubated for 30min at 4°C and washed twice with FACS buffer by spinning at 500G for 2min; 1% PFA (Paraformaldehyde) in PBS was added to cell pellet to fix the cells, followed by FACS analysis. VHH clones tested: 1B5: stock = 1.6 mg/ml; 1H1 : stock = 1.9 mg/ml; 1G6: stock = 1.1 mg/ml; 2F3: stock = 1.45 mg/ml; 2F5: stock = 0.5 mg/ml; 2H3: stock = 2mg/ml. As shown in Figures 2A-2C, binding of clones 1H1, 1G6, 2F5 and 2H3 to U937 cells was detected with anti-His-PE; very little binding of clones 1B5 and 2F3 to U937 cells could be detected by anti-His-PE.

[0122] U937 cell binding by VHH clones was assessed using varying doses to determine

EC50s. Briefly, the method included: VHH clones (3ug/ml as starting conc./test, with 3x serial dilutions) in FACS buffer were added to Fc blocked U937 or 293T-CLL1 cells (2.5c10 ^L 5), the mixtures were incubated for 30min at 4°C, then washed twice with FACS buffer by spinning at 500G for 2min; anti-His-PE was added to cells, the mixture was incubated for 30min at 4°C and washed twice with FACS buffer by spinning at 500G for 2min; 1% PFA (Paraformaldehyde) in PBS was added to cell pellet to fix the cells, followed by FACS analysis. VHH clones tested:

1B5: stock = 1.6 mg/ml; 1H1 : stock = 1.9 mg/ml; 1G6: stock = 1.1 mg/ml;2F3: stock = 1.45 mg/ml; 2F5: stock = 0.5 mg/ml; 2H3: stock = 2mg/ml. As shown in Figures 3A-3C, the EC50s of clones on U937 cells were: lG6=0.49nM, 2F5=0.47nM, 2H3=0.35nM and lHl=81nM.

[0123] Additional VHH clones were assessed for binding to E1937. Briefly, VHH clones

(3pg/ml as final conc./test) in FACS buffer were added to Fc blocked E1937 cells (2.5c10 ^L 5), the mixtures were incubated for 30min at 4°C, then washed twice with FACS buffer by spinning at 500G for 2min; anti-cMyc (Novus, Cat. # NBP2-37822) was added to cells, the mixture was incubated for 30min / 4°C and washed twice with FACS buffer by spinning at 500G / 2min, then anti-msIgG-PE (Jackson ImmunoResearch, Cat. # 115-116— 146) was added to the cells, the mixture was incubated for 30min / 4°C and washed twice with FACS buffer by spinning at 500G / 2min, 1% PFA (Paraformaldehyde) in PBS was added to cell pellet to fix the cells, followed by FACS analysis. Clones tested: 1A10: stock = 0.9 mg/ml; 1B1 : stock = 0.9 mg/ml; 1C6: stock = 1.5mg/ml; 2C8: stock = 0.9 mg/ml; 1D2: stock = 0.06 mg/ml. Previous clone as control 2F5: stock = 0.5 mg/ml. As shown in Figures 4A and 4B, four clones showed 99% binding on U937 by a-cMyc detection.

[0124] An additional plate was screened for clones binding to U937 cells. Briefly, 10m1 of clone lysate was added to 90m1 of Fc blocked U937 (2.5c10 ^L 5) in FACS buffer, incubated for 30min at 4°C, washed twice with FACS buffer by spinning at 500G for 2min, a-cMyc-PE (Novus Cat. # NB200-108PE, 1 :200 dilution) was added to the cells, incubated for 30min at 4°C, washed twice with FACS buffer by spinning at 500G for 2min, fixed with 1% PFA in PBS, then analyzed by flow cytometry. As shown in Figures 5A-5D, 31 anti-CLLl positive clones were identified from 96 lysates.

[0125] Additional plates were screened for clones binding to U937 cells. Briefly, 10m1 of clone lysate was added to 90m1 of Fc blocked E1937 (2.5c10 ^L 5) in FACS buffer, incubated for 30min at 4°C, washed twice with FACS buffer by spinning at 500G for 2min, a-cMyc-PE (1 :200 dilution) was added, incubated for 30min/4°C, washed twice with FACS buffer by spinning at 500G for 2min, fixed with 1% PFA in PBS, then analyzed by flow cytometry. As shown in Figures 5A-5D, 31 anti-CLLl positive clones were identified from 96 lysates from a first plate. As shown in Figures 6A-6C, 28 anti-CLLl positive clones were identified from 96 lysates from a second plate.

[0126] A summary of binding is provided in Table 3:

Table 3:

[0127] ELISAs were used to determine if the VHH clones recognized distinct or similar epitopes. 96 well plates (Pierce Cat. # 15041) were coated with 0. lug/ml soluble CLEC12a-His (Sino Biological # 11896-H07H) in 0.1 M carbonate buffer, pH9.5, O/N at 4C. The plate was blocked with 0.3% nonfat milk in TBS (200 ul/well) for 1 hr at RT. The plates were washed 3x with lxTBST (0.1M Tris, 0.5M NaCl and 0.05% Tween-20). The test anti-CLEC12a VHHs were added at 10 ug/ml horizontally across the plate, 100 ul per well and incubated for lh.

Without washing, the test anti-CLEC12a biotinylated VHHs were added at 0.2 ug/ml vertically down the plate, 100 ul per well and incubate for 1 h. After being washed 3x with lxTBST, HRP- conjugated streptavidin (Pierce #21130) at 1 :2000 (100 ul/well) was added, followed by incubation at RT in the dark for 1 hr. For detection, 1-Step Ultra TMB-ELISA reagent (Thermo Fisher #34028) was added at 100 ul per well and read at 450 nm. The mapping results indicated that most anti-CLEC12A VHHs recognized the same epitope, except for 1H1 and perhaps 2C2. This indicates that the VHHs from 1B1, IG6 and 2F5 recognize the same epitope as the VHHs from clones that differ in CDR1 (i.e., 1 A10 and 2H3). The IH1 VHH was distinct from these, and 2C2 was intermediate. Results are depicted in Figures 7A-7D.

Example 2 - Epitope mapping

[0128] One or more of the following methods are used to define the binding epitope for each antibody. Epitopes are either linear or conformational. A linear epitope is composed of a linear stretch of amino acids in the sequence that does not form 3 -dimensional structure. A conformational epitope is one that requires tertiary folding to create the proper binding region. Epitope mapping techniques are used for identifying either linear or conformational epitopes. However, linear techniques do not map conformational epitopes. Linear techniques include peptide array scanning, scanning mutagenesis, peptide library phage display and related techniques that define the amino acids found in the linear epitope. These linear techniques are used to map the epitope of antibodies that can bind to a non-conformational part of the antigen, e.g., as is known for antibodies that can bind to denatured proteins that have had their conformations disrupted. Most epitopes are conformational and are defined by using one or more techniques including X-ray co-crystallography, conformational peptide scanning as can be performed with large peptides displayed on the surface of phage, mutagenesis technologies whereby specific amino acid residues of the antigen are mutated or changed (often to alanine) and the presence of the complex is detected (often with fluorescence, e.g., in FRET assays), a technique that can be automated, i.e., as when many plasmid clones are generated in a library format using computers to perform statistical calculations of the database, cross-link coupled Mass Spectrometry in which the antibody and the antigen are tagged with a mass-labeled chemical crosslinker. The antibody/antigen complex is confirmed using high mass MALDI detection. Once created the antibody/antigen complex is extremely stable, and various enzymes and digestion conditions are applied to the complex to provide many different overlapping peptides. Identification of these peptides is performed using high-resolution mass spectrometry and MS/MS techniques. Identification of the crosslinked peptides is determined using mass tags linked to the cross-linking reagents. After MS/MS fragmentation and data analysis using specific interaction software, both epitope and paratope are determined in the same experiment. In another technique, Hydrogen Deuterium Exchange (HDX), the availability of hydrogen molecules in the backbone of a protein structure is measured. During analysis, both the unbound antigen and the bound antibody-antigen complex are incubated in deuterated water in order to exchange any hydrogens from exposed amino acids of the protein's backbone. By comparing the unbound antigen with the bound antibody-antigen complex the residues of the epitope are determined. One or more of these technologies is used to identify linear and conformational epitopes of the VHH antibodies of the invention.

Linear Epitopes

[0129] To evaluate if any identified VHH clones bind a linear epitope on Clecl2A, clones were evaluated for binding denatured Clecl2A via Western Blot following SDS-PAGE. To test recognition of Clecl2A by the VHH clones, 0.5 m of Clecl2A-His (Sino Biological,

11896-H07H) was run on a 4-12% gradient SDS gel (Invitrogen, NP0321) under reducing conditions. After blotting, using the iBlot 2 system (Invitrogen, IB23002), the membrane was blocked in 5% non-fat milk in Tris buffered saline (TBS) for 1 hr. Next, half the blot was incubated with about 1 ug/ml protein supernatant of myc tagged anti CLEC12a VHH for 2 hrs followed by 3x wash in wash buffer (lx TBST: 0.1 M Tris, 0.5 M NaCl, 0.05% Tween 20). The blot was then incubated with 1 :2000 HRP-anti-Myc (Rockland/Fisher, 50-105-8097) for another 1 hr and washed 3x in wash buffer and then developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher, 34095). The other half of the blot was directly incubated with 1 :2000 HRP-anti-his (BioLegend/Fisher, 652504) for 1 hr at 0.1 pg/ml and developed as a positive control for the presence of Clecl2A on the blot. Only one clone, VHH clone 1H1, was found to bind denatured Clecl2A by Western Blot. Results are depicted in Figure 8.

Example 3- VHH clones recognize an epitope distinct from previously reported anti-Clecl2A scFv

[0130] To further elucidate epitopes bound by the VHH clones, several VHH clones were tested for their ability to compete for binding with a known anti-Clecl2A scFv. A fusion protein comprising the anti-Clecl2A scFv SC02-357 described in US Patent 7741443 (included herein as SEQ ID NO:26) was immobilized on plastic plates followed by binding of recombinant Clecl2A. Three biotinylated VHH clones, 1A10, 1B1, and 2H3, each from the same epitope cluster, and a negative control of biotinylated Clecl2A (lane 1), were then evaluated for binding. Each of the three clones was able to bind the Clecl2A pre-bound to the anti-Clecl2A scFv. This result (Figure 9) demonstrates that the epitope recognized by the anti-Clecl2A scFv is distinct from the epitope recognized by the VHH clones.

Example 4 Binding of VHH clones to Clecl2A variants

[0131] The ability of VHH clones to recognize commercially available recombinant

Clecl2a proteins was tested by ELISA. Briefly, a 96 well plate (Pierce, 15041) was coated with l.O ug/ml Clecl2A (Sino Biological, 11896-H07H or ABclonal, RP01018) in 0.1 M carbonate, pH9.5 overnight at 4°C. The plate was blocked with 200 mΐ/well 0.3% non-fat milk in Tris buffered saline (TBS) for 1 hr at room temperature (RT). Then, the plates were washed 3x with wash buffer (lx TBST: 0.1 M Tris, 0.5 M NaCl, 0.05% Tween 20). Next, 100 mΐ of the 2H3 VHH-Myc-His (SEQ ID NO: 15, purified by ProSci), was added starting at 5 pg/ml with 3 fold dilutions, and incubated for lh at RT. The plate was washed 3x with wash buffer and 100 mΐ of 1 ug/ml anti-Myc antibody (NOVUS/Fisher, NBP2-37822) was added per well. The plate was incubated for lh at RT and then washed 3x with wash buffer. Following the wash step, 100 mΐ of a 1 :2000 dilution of HRP-anti-mlgG (Jackson ImmunoRe search, 115-035-062) was added per well and incubated at RT in the dark for 1 hr. For detection, 100 mΐ of 1-Step Ultra TMB-ELISA from Thermo Fisher, 34028 was added per well and then the plate was read at 405 nM when the color developed. This experiment was repeated twice. Graphs for each experiment were generated using the Softmax software and are shown in Figure 10A.

[0132] In a second ELISA, the plate was coated with the Clecl2A proteins as above, but instead of probing with the 2H3 VHH, a 2H3 VHH-G4Sx4-CD19-His fusion protein was added starting at 5 pg/ml with 3 fold dilutions. Also, instead of the anti-Myc reagent, the anti-CD 19 Ab FMC63 was added at 1 pg/ml cone. After washing, the protocol using the HRP anti-mlgG reagent was followed as above. Graphs were generated using the Softmax software and are shown in Figure 10B.

[0133] It is understood that commercially available recombinant Clecl2A proteins have an added six histidine tag to allow for easy analysis. The Clecl2A from Sino Biological has the HIS tag at the N-terminus, while the Clecl2A from ABclonal has the HIS tag at the C-terminus. We found that VHH clone 2H3 bound immobilized Sino Biological Clecl2A but not immobilized ABclonal Clecl2A (see Figure 10A). It is possible that the epitope for the VHH cluster containing 2H3 bound close to the C-terminus, i.e. that the added C-terminal HIS tag might be interfering with binding. It should be noted that Clecl2A is a Type II membrane protein, i.e. the C-terminus is extracellular. To obviate potential problems with direct binding of the Clecl2A to plastic (Figure 4A), the Clecl2A’s from each source were also immobilized using an anti-Clecl2A antibody immobilized on plastic plates first, which gave similar results (Figure 10B). [0134] To further investigate whether the observed difference in binding was caused by the HIS tag at the C-terminus interfering with binding of clone 2H3 we reviewed the amino acid sequences of the two recombinant Clecl2A proteins. We discovered that there are two forms of Clecl2A with a single amino acid difference in this region. The canonical sequence (UniProt; Q5QGZ9-1) contains a Lysine (K in bold underline; SEQ ID NO:27 ).

[0135] MWIDFF T YS SM SEE VT Y ADLQF QN S SEMEKIPEIGKF GEK APP AP SH VWR

PAALFLTLLCLLLLIGLGVLASMFHVTLKIEMKKMNKLQNISEELQRNISLQLMSNM NIS NKIRNLSTTLQTIATKLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLSDDVQTWQESKM ACAAQNASLLKINNKNALEFIKSQSRSYDYWLGLSPEEDSTRGMRVDNIINSSAWVIRN APDLNNMYCGYINRLYVQYYHCTYKKRMICEKMANPVQLGSTYFREA (SEQ ID NO:

27)

[0136] However, the Sino Biological sequence (GenBank: EAW96132.1) has a

Glutamine instead (Q in in bold underline; SEQ ID NO:28).

[0137] MWIDFF T YS SM SEE VT Y ADLQF QN S SEMEKIPEIGKF GEK APP AP SH VWR

PAALFLTLLCLLLLIGLGVLASMFHVTLKIEMKKMNKLQNISEELQRNISLQLMSNM NIS

NKIRNLSTTLQTIATKLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLSDDVQTWQES KM

ACAAQNASLLKINNKNALEFIKSQSRSYDYWLGLSPEEDSTRGMRVDNIINSSAWVI RN

APDLNNMYCGYINRLYVQYYHCTYKQRMICEKMANPVQLGSTYFREA (SEQ ID

NO:28)

[0138] Upon evaluation of binding to other Clecl2A sequences we found that VHH clone 2H3 did not bind a Clecl2A from AbClonal (NCBI Reference Sequence:

NP OOl 193939.1, isoform3) or a cDNA from Genscript both of which contain a Lysine residue at amino acid 254. This suggests that the K/Q amino-acid is within or close to the 2H3 epitope, and is consistent with the 2H3 epitope being close to the C-terminus. In contrast the scFv binds to both protein variants.

Example 5- Mass Spectrometry based Epitope Identification

[0139] To further define the epitopes of the scFv and VHH 2H3, we used a commercially available process (CovalX), which covalently cross-links the binders to the target, then uses mass spectrometry to define the binding sites. By mixing a protein sample containing non-covalent interactions with a specially developed cross-linking mixture (Bich, C et al. Anal. Chem., 2010, 82 (1), pp 172-179), it is possible to specifically detect non-covalent complex with high- sensitivity. The covalent binding generated allows the interacting species to survive the sample preparation process and the MALDI ionization. A special High-Mass detection system allows characterizing the interaction in the High-Mass range. Figure 11 A shows the amino acids crosslinked between 2H3 and Clecl2A defining the region of the 2H3 epitope. Amino acids 251 (T); 252 (Y); and 260 (K) (based on full length Clecl2A SEQ ID NO 28) were identified as cross linked. The epitope spans the region containing the K/Q variant, entirely consistent with a change in charge in this region (K vs. Q) disrupting the 2H3 epitope as suggested in Example 3. Figure 1 IB shows the epitope for the SC02-357 scFv. Again, its presence near the C-terminal is consistent with all other results, i.e., a distinct epitope from 2H3, yet within the last 50 AA of Clecl2A.

Example 6- Specificty of Binding

[0140] To examine binding to proteins related to Clecl2A, family members with the highest degree of homology were determined. CleclA and Clecl2B were the most related with identity in their extracellular domains of 36% and 32%, respectively. Expression plasmids for C terminal Flag tagged Clecl2A (OHu09814D), Clecl2B (OHul3983B) and CleclA

(OHu27138D) were purchased from GenScript. They were transfected into 293T cells using 2 pg plasmid and 10 mΐ of lipofectamine 2000 (Invitrogen, 11668019) per well (6 well plate) using the manufacturer’s protocol. After 48 hrs, the cells were removed using Accutase (Thermo Fisher, 00-4555-56) and washed with PBS. The cells were then blocked for 10 minutes with human Fc- Block (BD Biosciences, 564219). Fc-blocked 293T-Clecl2A; Clecl2B; or CleclA cells were resuspended in 50 mΐ FACs buffer (PBS / 1% BSA / 0.1% Sodium Azide) and then added to 50 mΐ of a 2H3VHH-CD19-His fusion protein (His Trap Excel (GE Healthcare, 17-3712 purified) serially diluted in FACS buffer, starting at 5 pg/ml, final concentration. The sequence of the 2H3 VHH used is a version of SEQ ID NO: 15 lacking the C-terminal myc and His tags disclosed in SEQ ID 29Serial dilutions were done in 3-fold steps. The cells and the 2H3VHH-CD19 fusion protein were incubated together for 30 minutes at 4°C. The samples were washed in FACS buffer and centrifuged at 500 RCF at 4°C for 2 minutes. This wash step was repeated and then the cells were resuspended in FACs buffer containing 2.5 pg/ml PE-labeled FMC63 (MilliporeSigma, MAB1794H). After incubation for 30 minutes at 4°C, the cells were washed twice as described resuspended in 150 mΐ PBS / 1% paraformaldehyde to fix the cells. Expression of the Clec receptors was verified by staining the transfected cells with 5 mΐ anti-Flag-APC reagent (BioLegend, 637307) per sample. After 30 minutes at 4°C, the samples were washed and fixed as above. Cell samples data were collected by flow cytometry (BD Accuri 6) and analyzed using BD Accuri 6 software. It was determined that the fusion protein containing the VHH 2H3 only bound to Clecl2A expressing cells (Figure 12).

Other Embodiments

[0141] While a number of embodiments of this invention are described herein, the present disclosure and examples may be altered to provide other methods and compositions of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims in addition to the specific embodiments that have been represented by way of example. All references cited herein are hereby incorporated by reference.

LISTING OF SEQUENCES

SEP ID NO:l

atgtctgaag aagttactta tgcagatctt caattccaga actccagtga gatggaaaaa 60 atcccagaaa ttggcaaatt tggggaaaaa gcacctccag ctccctctca tgtatggcgt 120 ccagcagcct tgtttctgac tcttctgtgc cttctgttgc tcattggatt gggagtcttg 180 gcaagcatgt ttcatgtaac tttgaagata gaaatgaaaa aaatgaacaa actacaaaac 240 atcagtgaag agctccagag aaatatttct ctacaactga tgagtaacat gaatatctcc 300 aacaagatca ggaacctctc caccacactg caaacaatag ccaccaaatt atgtcgtgag 360 ctatatagca aagaacaaga gcacaaatgt aagccttgtc caaggagatg gatttggcat 420 aaggacagct gttatttcct aagtgatgat gtccaaacat ggcaggagag taaaatggcc 480 tgtgctgctc agaatgccag cctgttgaag ataaacaaca aaaatgcatt ggaatttata 540 aaatcccaga gtagatcata tgactattgg ctgggattat ctcctgaaga agattccact 600 cgtggtatga gagtggataa tataatcaac tcctctgcct gggttataag aaacgcacct 660 gacttaaata acatgtattg tggatatata aatagactat atgttcaata ttatcactgc 720 acttataaac aaagaatgat atgtgagaag atggccaatc cagtgcagct tggttctaca 780 tattttaggg aggcatgagg c 801

SEP ID NO:2

Met Ser Glu Glu Val Thr Tyr Ala Asp Leu Gin Phe Gin Asn Ser Ser 1 5 10 15

Glu Met Glu Lys lie Pro Glu lie Gly Lys Phe Gly Glu Lys Ala Pro 20 25 30

Pro Ala Pro Ser His Val Trp Arg Pro Ala Ala Leu Phe Leu Thr Leu 35 40 45

Leu Cys Leu Leu Leu Leu lie Gly Leu Gly Val Leu Ala Ser Met Phe His Val Thr Leu Lys lie Glu Met Lys Lys Met Asn Lys Leu Gin Asn

65 70 75 80 lie Ser Glu Glu Leu Gin Arg Asn lie Ser Leu Gin Leu Met Ser Asn

85 90 95

Met Asn lie Ser Asn Lys lie Arg Asn Leu Ser Thr Thr Leu Gin Thr

100 105 110

lie Ala Thr Lys Leu Cys Arg Glu Leu Tyr Ser Lys Glu Gin Glu His

115 12 0 125

Lys Cys Lys Pro Cys Pro Arg Arg Trp lie Trp His Lys Asp Ser Cys

130 135 14 0

Tyr Phe Leu Ser Asp Asp Val Gin Thr Trp Gin Glu Ser Lys Met Ala

145 15 0 155 160

Cys Ala Ala Gin Asn Ala Ser Leu Leu Lys lie Asn Asn Lys Asn Ala

165 17 0 175

Leu Glu Phe lie Lys Ser Gin Ser Arg Ser Tyr Asp Tyr Trp Leu Gly

18 0 185 190

Leu Ser Pro Glu Glu Asp Ser Thr Arg Gly Met Arg Val Asp Asn lie

195 200 205

lie Asn Ser Ser Ala Trp Val lie Arg Asn Ala Pro Asp Leu Asn Asn

210 215 22 0

Met Tyr Cys Gly Tyr lie Asn Arg Leu Tyr Val Gin Tyr Tyr His Cys

225 230 235 24 0

Thr Tyr Lys Lys Arg Met lie Cys Glu Lys Met Ala Asn Pro Val Gin

245 25 0 255 Leu Gly Ser Thr Tyr Phe Arg Glu Ala

2 60 2 65

SEQ ID NO:3 (underlining denotes CDR 1. CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids ( TSGPGGQGA ), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS))

OVOLOESGGGLVOAGGSLRLSCAASGSIFAINEINLMGWYROAPGKORELVAACASDG NT YY AD S VKGRFTISRDNAEKT VYLOMNNLKPDDT AVYY CD AN SRGNYYSGOGT 0 VI V S S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 4 (underlining denotes CDRE CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS))

OVOLOESGGGLVOAGGSLRLSCAASGSIFAINEINLMGWYROAPGKORELVAACASDG NT YY AD S VKGRFTISRDNAEKT VYLOMNNLKPDDT AVYY CD AN SRGNYYSGOGTL VT VS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 5 (underlining denotes CDRE CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS))

OVOLOOSGGGLVOAGGSLRLSCAASGSIFAINEINLMGWYROAPGKORELVAACASDG NT YY AD S VKGRFTISRDNAEKT VYLOMNNLKPDDT AVYY CD AN SRGNYYSGOGT O VT VS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 6 (underlining denotes CDRE CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLOOSGGGLVOAGGSLRLSCAASGSIFAINEINLMGWYROAPGKORELVAACASDG NT YY AD S VKGRFTISRDNAEKT VYLOMNNLKPDDT AVYY CD AN SRGNYYSGOGTL VT VS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS SEQ ID NO: 7 (underlining denotes CDRL CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLOOFGGGLVOAGGSLRLSCAASGSIFAINEINLMGWYROAPGKORELVAACASDG NT YY AD S VKGRFTISRDNAEKT VYLOMNNLKPDDT AVYY CD AN SRGNYYSGOGTL VT VS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 8 (underlining denotes CDR 1. CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids ( TSGPGGQGA ), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLOASGGGLVOAGGSLRLSCAASGSIFAINEINLMGWYROAPGKORELVAACASDG NT YY AD S VKGRFTISRDNAEKT VYLOMNNLKPDDT AVYY CD AN SRGNYYSGOGT O VT VS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 9 (underlining denotes CDRL CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLOEFGGGLVOAGGSLRLSCAASGSIFAINEINLMGWYROAPGKORELVAACASDG NT YY AD S VKGRFTISRDNAEKT VYLOMNNLKPDDT AVYY CD AN SRGNYYSGOGTL VT VS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 10 (underlining denotes CDR 1. CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLOEFGGGLVOAGGSLRLSCAASGSIFAINEINLMGWYROAPGKORELVAACASDG NT YY AD S VKGRFTISRDNAEKT VYLOMNNLKPDDT AVYY CD AN SRGNYYSGOGT O VT VS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS SEQ ID NO: 11 (underlining denotes CDRE CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLVESGGGLVOAGGSLRLSCAASGSIFAINEINLMGWYROAPGKORELVAACASDG NT YY AD S VKGRFTISRDNAEKT VYLOMNNLKPDDT AVYY CD AN SRGNYYSGOGTLVT VS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 12 (underlining denotes CDR 1. CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids ( TSGPGGQGA ), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLOOSGGGLAOTGGSLILSCAASGSIFAINEINLMGWYROAPGKORELVAACASDGN TYYADSVKGRFTISRDNAEKTVYLOMNNLKPDDTAVYYCDANSRGNYYSGOGTOVTV S S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 13 (underlining denotes CDR 1. CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLOESGGGLVOPGGSLRLSCAASGSIFAINEINLMGWYROAPGKORELVAACASDG NT YY AD S VKGRFTISRDNAEKT VYLOMNNLKPDDT AVYY CD AN SRGNYYSGOGTLVT VS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 14 (underlining denotes CDR 1. CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

Q V QLQES GGGL V Q VGE SLRL S CVVSGDTRSINLMGW YRQ APGKQREL VAACASDGNT YYADS VKGRFTISRDNAEKTVYLOMNNLKPDDTAVYY CD AN SRGNYYSGOGTLVT V S S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 15 (underlining denotes CDRE CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag (EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OYOLOESGGGLYOAGGSLRLSCVASGSiRSINVMGWYROAPGKORELVAACASDGNTY Y AD S VKGRFTISRDNAEKT VYLOMNNLKPDDT AVYY CD AN SRGNYYSGOGT O VT V S S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 16 (underlining denotes CDR 1. CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids ( TSGPGGQGA ), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLOESGGGLVOPGGSLRLSCAASGFTFNSYAMTWVROAPGKGLEWVSDINSGGGST NY AD S VKGRF TI SRDNAKNTL YLOMN SLKPEDT A V Y Y C ATELRGSD Y YRGPIRE Y A YW GQGTL VT VS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 17 (underlining denotes CDRE CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLOESGGALVOAGGSLRLSCAASGLTFSNYAMGWFROAPGKEREFVAAINWSGGT TDYATSVKGRFTISRDNAKNTVYLOLNSLKPEDTAVYYCAASYRLRITVVVTPDEYHY WGQGTLVTVS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 18 (underlining denotes CDRE CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLOOSGGGLVOPGGSLRLSCAASGFAFDDYAMIWVROGPGKGLEWVSSISWNGGG TYYAESIVGRFTVSRDNAKKMVYLOMNGLKSEDTAMYYCVKLVDSGWYSAYDYWGO GTQVTVS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO: 19 (underlining denotes CDRE CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) : OVOLOESGGGLVOAGGSLRLSCVVSGATSNVNAMGWYROAPGKERELVAAISSGGSTS YRDSVKGRFTISRDNAKNTLYLOMNSLKPEDTAMYYCAAODWATEGYEYDYWGOGT L VT VS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO:20 (underlining denotes CDR 1. CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids ( TSGPGGQGA ), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

O VOLO AFGGGL VOPGGSLRLSC VVSGTMF SGKDVNWLROAPGKHVEVVAT VS SDGGT D Y ADF VKGRFTISRDD AKNT VNLOMN SLEPEDT ANYMCHFLW GRHYW GOGT O VT V S S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO:21 (underlining denotes CDRE CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLOAFGGGMVOAGESLRLSCVASGNDISGSAMAWYRAHLGAERELVAVDAPRERP

FYIDPVIGRFTISRDDRNKMLYLOMNDLRPDDTATYWCGPSLRTFHGREWYRPPWFT S

WGQGTQYTYSSTSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO:22 (underlining denotes CDR 1. CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

OVOLOOSGGGLVOAGGSLRLSCAASGSIFSINAMGWYROAPGKRREMVAVVSRFGETT YT GS VKGRFTISRINKN1NT VFLOMNRLKPEDT AVYY CNARIRGNY GSRID YW GOGT O V TVSS TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO:23 (underlining denotes CDRE CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) : OVOLOEFGGGLVOLGGSARLSCVVSGNMLDLNTMAWYROGELVAALGISTYYAESVK GRFTISRDNAKNTLYLQMN SLKSEDT AVYY C ARDYNFESW GQGTL VT V S S TSGPGGQG AEQKLISEEDLGAHHHHHHGAS

SEQ ID NO:24 (underlining denotes CDR 1. CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids ( TSGPGGQGA ), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

AVOLOESGGGLVOAGGSLRLSCAASGSDRSINVMNWYROAPGKORELVAAITSGGTTN Y AO S VKGRVTISRD S AKNT VYLOMN SLKPEDT AVYF CK ADTRW GGMYW GPGTO VT V S S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO:25 (underlining denotes CDRE CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes (i) a linker of 9 amino acids (TSGPGGQGA), (ii) a myc-tag

(EQKLISEEDL), (iii) a linker of 2 amino acids (GA), (iv) a hexa-histidine tag (HHHHHH), and (v) an additional 3 amino acids (GAS)) :

O V OLOO S GGGL V O AGGSLTL S C A AT GRTIDN G AM AWFRO APGKOREL V A AINW S GGA TFYTDSVKYRFTISRDNVRHTLDLOMTSLKPEDTTIYFCASRRGVDLRRNSYEYDYWGR GTLVT VS S TSGPGGQGAEQKLISEEDLGAHHHHHHGAS

SEQ ID NO:26:

QVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSPD YNP SLK SR VTI S VDK SRN QF SLKL S S VT A ADT A V Y Y C AK V S T GGFFD YW GQGTL VTV S S GGGGSGGGGSGGGGSEIELTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPK LLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGPGTKVE IK

SEQ ID NO: 27

MWIDFFTYSSMSEEVTYADLQFQNSSEMEKIPEIGKFGEKAPP APSHVWRP AALFLTLLC LLLLIGLGVLASMFHVTLKIEMKKMNKLQNISEELQRNISLQLMSNMNISNKIRNLSTTL QTIATKLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLSDDVQTWQESKMACAAQNASL LKINNKNALEFIKSQ SRS YD YWLGL SPEED STRGMRVDNIIN S SAW VIRNAPDLNNM Y C GYINRL YVQYYHCTYKKRMICEKMANPVQLGSTYFREA

SEQ ID NO:28.

MWIDFFT Y S SMSEE VT Y ADLQF QN S SEMEKIPEIGKF GEK APP APSHVWRP AALFLTLLC LLLLIGLGVLASMFHVTLKIEMKKMNKLQNISEELQRNISLQLMSNMNISNKIRNLSTTL QTIATKLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLSDDVQTWQESKMACAAQNASL LKINNKNALEFIKSQ SRS YD YWLGL SPEED STRGMRVDNIIN S S AW VIRNAPDLNNM Y C GYINRL Y V Q YYHC T YKQRMICEKM ANP V QLGS T YFRE A SEQ ID NO:29 (underlining denotes CDR 1. CDR2. CDR3. sequentially; bolded italics at C- ter minus denotes a linker of 9 amino acids (TSGPGGQGA)

OYOLOESGGGLVOAGGSLRLSCVASGSiRSINVMGWYROAPGKORELVAACASDGNTY Y AD S VKGRFTISRDNAEKT VYLOMNNLKPDDT AVYY CD AN SRGNYYSGOGT O VT V S S TSGPGGQGA