Single-domain antibodies against CLL1 and constructs thereof

Abstract

Provided are single-domain antibodies targeting CLL1 and constructs thereof, including chimeric receptors, immune effector cell engagers and immunoconjugates. Further provided are engineered immune effector cells (such as T cells) comprising an anti-CLL1 chimeric receptor and optionally a second chimeric receptor targeting a second antigen or epitope. Pharmaceutical compo- sitions, kits and methods of treating cancer are also provided.

Description

SINGLE-DOMAIN ANTIBODIES AGAINST CLL1 AND CONSTRUCTS THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefits of International Patent Application No. PCT / CN2018 / 104883 filed September 10, 2018, and International Patent Application No. PCT / CN2018 / 104882 filed September 10, 2018.

[0002] FIELD OF THE PRESENT APPLICATION

[0003] The present invention relates to single-domain antibodies, chimeric receptors and engineered immune cells that target CLL1, and methods of use thereof. BACKGROUND OF THE PRESENT APPLICATION

[0004] With the development of tumor immunotherapy and clinical technology, chimeric antigen receptor T cell (CAR-T) immunotherapy is now one of the most promising tumor immunotherapy approaches. Generally, a chimeric antigen receptor (CAR) comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain. The extracellular domain may comprise a single chain variable fragment (scFv) targeting an identified tumor antigen. CARs can be expressed on the surface of T cells using gene transfection techniques. Upon binding to the target tumor antigen, the CARs can activate the T cells to launch specific anti-tumor response in an antigen-dependent manner without being limited by the availability of major histocompatibility complexes (MHC) specific to the target tumor antigen.

[0005] Single-domain antibodies (sdAbs) are different from conventional 4-chain antibodies by having a single monomeric antibody variable domain. For example, camelids and sharks produce sdAbs named heavy chain-only antibodies (HcAbs), which naturally lack light chains. The antigen-binding fragment in each arm of the camelid heavy-chain only antibodies has a single heavy chain variable domain (VHH), which can have high affinity to an antigen without the aid of a light chain. Camelid VHH is known as the smallest functional antigen-binding fragment with a molecular weight of approximately 15 kD.

[0006] Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells, characterized by the rapid growth of immature blood cells ("blasts") that build up in the bone marrow and blood and interfere with normal blood cells. AML may spread to other organs, such as the liver, spleen, and brain. Clinical symptoms of AML include feeling tired, shortness of breath, easy bruising and bleeding, and increased risk of infection. Without treatment, AML progresses rapidly and is typically fatal within weeks or months. AML has several subtypes for which treatments and outcomes may vary. Typically, AML is initially treated with chemotherapy, sometimes along with a targeted therapy drug. Patients may then go on to receive a stem cell transplant, additional chemotherapy, surgery, or radiation therapy. AML most commonly occurs in older adults, some of whom are not healthy enough to receive intensive chemotherapy and thus have poor clinical outcome. Although current therapies for AML often lead to remissions, almost all patients eventually relapse. There is a need for an effective immunotherapeutic agent to treat AML.

[0007] BRIEF SUMMARY OF THE PRESENT APPLICATION

[0008] The present application provides anti-CLL1 single-domain antibodies (sdAb) and constructs thereof, including chimeric receptors, immune effector cell engagers, and immunoconjugates, engineered immune cells, and methods of use thereof in cancer immunotherapy.

[0009] One aspect of the present application provides an anti-CLL1 construct comprising an single domain antibody ("sdAb") moiety that specifically binds to CLL1, wherein the sdAb moiety (e.g., VHH) comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, 74, 81, 88, 151, 158 and 165, or a variant thereof comprising up to about 3 amino acid substitutions in the CDR1; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 6, 13, 20, 27, 34, 41, 48, 55, 62, 69, 76, 83, 90, 153, 160, and 167, or a variant thereof comprising up to about 3 amino acid substitutions in the CDR2; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 155, 162, and 169, or a variant thereof comprising up to about 3 amino acid substitutions in the CDR3. In some embodiments, the sdAb moiety comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 57, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60, a CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 64, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO: 74, a CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 78, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO: 81, a CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 85, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a CDR2 comprising the amino acid sequence of SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 92, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO: 151, a CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 155, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO: 158, a CDR2 comprising the amino acid sequence of SEQ ID NO: 160, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 162, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; or (16) a CDR1 comprising the amino acid sequence of SEQ ID NO: 165, a CDR2 comprising the amino acid sequence of SEQ ID NO: 167, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 169, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs.

[0010] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1, a CDR2, and a CDR3 of an sdAb comprising the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173.

[0011] In some embodiments according to any one of the anti-CLL1 constructs described herein, the sdAb moiety comprises an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171- 173. In some embodiments, the sdAb moiety comprises the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173.

[0012] Also provided are CLL1 epitopes that any one of the anti-CLL1 sdAb moieties described above specifically bind to, and anti-CLL1 antibodies (such as anti-CLL1 sdAbs) that compete with any one of the anti-CLL1 sdAb moieties described above.

[0013] In some embodiments according to any one of the anti-CLL1 constructs described above, the sdAb moiety is a camelid antibody. In some embodiments, the sdAb moiety is a chimeric antibody. In some embodiments, the sdAb moiety is humanized. In some embodiments, the sdAb moiety is a VHH fragment.

[0014] In some embodiments, the anti-CLL1 construct is a chimeric receptor (also referred herein as "anti-CLL1 chimeric receptor") comprising an extracellular domain comprising the sdAb moiety (e.g., VHH), a transmembrane domain, and an intracellular signaling domain comprising a primary intracellular signaling sequence of an immune effector cell (e.g., T cell). In some embodiments, the primary intracellular signaling sequence is derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the primary intracellular signaling sequence is a CD3\(\zeta\) intracellular signaling sequence. In some embodiments, the intracellular signaling domain further comprises an intracellular co-stimulatory sequence. In some embodiments, the intracellular co-stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In some embodiments, the intracellular co-stimulatory sequence is derived from CD28 or 4-1BB. In some embodiments, the extracellular domain of the anti- CLL1 construct further comprises a second binding moiety that specifically binds to a second antigen or epitope. In some embodiments, the second binding moiety is an sdAb (e.g., VHH) or an scFv. In some embodiments, the second binding moiety is an extracellular domain of a receptor. In some embodiments, the second binding moiety specifically binds to CD33, CD123 or an NKG2D ligand. In some embodiments, the second binding moiety is an anti-CD33 sdAb or an anti-CD123 sdAb. In some embodiments, the second binding moiety is an extracellular domain (ECD) of NKG2D.

[0015] In some embodiments, the anti-CLL1 construct is a chimeric receptor (also referred herein as "anti-CLL1 chimeric receptor") comprising an extracellular domain comprising the sdAb moiety (e.g., VHH), a transmembrane domain, and an intracellular signaling domain comprising an intracellular co- stimulatory sequence. In some embodiments, the intracellular co-stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In some embodiments, the intracellular co-stimulatory sequence is derived from CD28 or 4-1BB. In some embodiments, the extracellular domain of the anti-CLL1 construct further comprises a second binding moiety that specifically binds to a second antigen or epitope. In some embodiments, the second binding moiety is an sdAb or an scFv. In some embodiments, the second binding moiety is an extracellular domain of a receptor. In some embodiments, the second binding moiety specifically binds to CD33, CD123 or an NKG2D ligand. In some embodiments, the second binding moiety is an sdAb or scFv that specifically binds to CD33 or CD123. In some embodiments, the second binding moiety is an ECD of NKG2D.

[0016] One aspect of the present application provides an anti-CLL1 chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb moiety, a transmembrane domain, and an intracellular signaling domain, wherein the anti-CLL1 sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling sequence of an immune effector cell (e.g., T cell). In some embodiments, the primary intracellular signaling sequence is derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the primary intracellular signaling sequence is a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence. In some embodiments, the intracellular signaling domain further comprises an intracellular co-stimulatory sequence. In some embodiments, the intracellular co-stimulatory sequence is derived from a co- stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In some embodiments, the intracellular co-stimulatory sequence is derived from CD28 or 4-1BB.

[0017] One aspect of the present application provides a multispecific chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb moiety and an anti-CD33 sdAb moiety, a transmembrane domain, and an intracellular signaling domain, wherein the anti-CLL1 sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43, and wherein the anti-CD33 sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 198, a CDR2 comprising the amino acid sequence of SEQ ID NO: 200, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 202. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling sequence of an immune effector cell (e.g., T cell). In some embodiments, the primary intracellular signaling sequence is derived from CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>, FcR<semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the primary intracellular signaling sequence is a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence. In some embodiments, the intracellular signaling domain further comprises an intracellular co-stimulatory sequence. In some embodiments, the intracellular co-stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In some embodiments, the intracellular co-stimulatory sequence is derived from CD28 or 4-1BB.

[0018] One aspect of the present application provides an anti-CLL1 chimeric receptor comprising the amino acid sequence of any one of SEQ ID NOs: 120-132, 177-179, 181, 184-195, and 229-230.

[0019] One aspect of the present application provides an engineered immune cell comprising any one of the anti-CLL1 chimeric receptors or multispecific chimeric receptors described above, or a nucleic acid encoding the anti-CLL1 chimeric receptor, or the multispecific chimeric receptor. In some embodiments, the engineered immune cell further comprises a second chimeric receptor. In some embodiments, the second chimeric receptor comprises an extracellular domain comprising a second binding moiety that specifically binds to a second antigen or epitope, a transmembrane domain, and an intracellular signaling domain comprising a primary intracellular signaling sequence of an immune effector cell (e.g., T cell). In some embodiments, the primary intracellular signaling sequence is derived from CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>, FcR<semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>, FcR<semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the primary intracellular signaling sequence is a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence. In some embodiments, the intracellular signaling domain further comprises an intracellular co-stimulatory sequence. In some embodiments, the second chimeric receptor comprises an extracellular domain comprising a second binding moiety that specifically binds to a second antigen or epitope, a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence. In some embodiments, the intracellular co-stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In some embodiments, the intracellular co-stimulatory sequence is derived from CD28 or 4-1BB. In some embodiments, the second binding moiety is an extracellular domain of a receptor. In some embodiments, the second binding moiety specifically binds to CD33, CD123 or an NKG2D ligand. In some embodiments, the second binding moiety is an sdAb or scFv that specifically binds to CD33 or CD123. In some embodiments, the second binding moiety is an ECD of NKG2D.

[0020] One aspect of the present application provides a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb, a transmembrane, and an intracellular signaling domain, wherein the anti-CLL1 sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8; or (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and (b) a second chimeric receptor comprising an extracellular domain comprising an anti-CD33 sdAb, a transmembrane domain, and an intracellular signaling domain, wherein the anti-CD33 sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR2 comprising the amino acid sequence of SEQ ID NO: 207, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 209; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 212, a CDR2 comprising the amino acid sequence of SEQ ID NO: 214, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 216; or (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 219, a CDR2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223. In some embodiments, the intracellular signaling domain of the first chimeric receptor and / or the second chimeric receptor comprises a primary intracellular signaling sequence of an immune effector cell (e.g., T cell). In some embodiments, the primary intracellular signaling sequence is derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the primary intracellular signaling sequence is a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence. In some embodiments, the intracellular signaling domain of the first chimeric receptor and / or the second chimeric receptor further comprises an intracellular co-stimulatory sequence. In some embodiments, the intracellular co-stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In some embodiments, the intracellular co-stimulatory sequence is derived from CD28 or 4-1BB. In some embodiments, the intracellular co-stimulatory sequence of the first chimeric receptor comprises an intracellular co-stimulatory sequence derived from CD28. In some embodiments, the intracellular co-stimulatory sequence of the second chimeric receptor comprises an intracellular co-stimulatory sequence derived from 4-1BB.

[0021] Also provided is a dual chimeric receptor construct comprising the amino acid sequence of any one of SEQ ID NOs: 234-236.

[0022] In some embodiments, there is provided an engineered immune cell comprising any one of the dual chimeric receptor systems or dual chimeric receptor constructs described above.

[0023] In some embodiments according to any one of the engineered immune cells described above, the immune cell is a T cell, an NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, or an embryonic stem cell. In some embodiments, the immune cell is a T cell, such as a cytotoxic T cell, a helper T cell, a natural killer T cell, or a γδT cell. In some embodiments, the engineered immune cell expresses a safety-switch antigen or epitope, such as CD52, EGFR, CD20 or an epitope thereof.

[0024] In some embodiments, the anti-CLL1 construct is a monospecific molecule. In some embodiments, the anti-CLL1 construct is a multispecific molecule, such as a bispecific molecule. In some embodiments, the anti-CLL1 construct is a secreted molecule. In some embodiments, the anti-CLL1 construct comprises the sdAb moiety (e.g., VHH) linked to a second binding moiety that specifically binds to a second antigen or epitope. In some embodiments, the second binding moiety is an sdAb or an scFv. In some embodiments, the sdAb moiety is linked to the second binding moiety via a peptide linker.

[0025] In some embodiments, the anti-CLL1 construct is an immune effector cell engager, wherein the second binding moiety specifically binds to an antigen on the surface of an immune cell. In some embodiments, the anti-CLL1 construct comprises a second binding moiety that specifically binds to an antigen on the surface of a T cell. In some embodiments, the second binding moiety specifically binds to an antigen is selected from the group consisting of CD3γ, CD3δ, CD3ε, CD3ζ, CD28, OX40, GITR, 4- 1BB, CD27, CD40L, and HVEM.

[0026] In some embodiments, the anti-CLL1 construct is an immunoconjugate comprising the sdAb moiety and an effector molecule. In some embodiments, the effector molecule is a therapeutic agent selected from the group consisting of a drug, a toxin, a radioisotope, a protein, a peptide, and a nucleic acid. In some embodiments, the effector molecule is a drug or a toxin. In some embodiments, the effector molecule is a label.

[0027] One aspect of the present application provides an isolated nucleic acid comprising a nucleic acid sequence encoding any one of the anti-CLL1 constructs (including anti-CLL1 sdAbs, anti-CLL1 chimeric receptors, multispecific chimeric receptors, dual chimeric receptor systems, immune effector cell engagers and anti-CLL1 immunoconjugates) described above. In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 107-119 and 174-176.

[0028] In some embodiments, there is provided an isolated nucleic acid comprising a nucleic acid sequence encoding any one of the anti-CLL1 chimeric receptors described above. In some embodiments, the isolated nucleic acid comprises a first nucleic acid sequence encoding the anti-CLL1 chimeric receptor, and a second nucleic acid sequence encoding a second chimeric receptor (e.g., anti-CD33, anti- CD123, or NKG2D chimeric receptor), wherein the second nucleic acid sequence is operably linked to the first nucleic acid sequence via a third nucleic acid sequence encoding a self-cleaving peptide, such as a T2A, P2A, or F2A peptide. In some embodiments, the isolated nucleic acid further comprises a nucleic acid sequence encoding a safety-switch antigen or epitope, such as CD52, CD20, EGFR or an epitope thereof.

[0029] One aspect of the present application provides a vector comprising any one of the isolated nucleic acids described above. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector, such as a lentiviral vector. In some embodiments, the vector is a non-viral vector.

[0030] One aspect of the present application provides a pharmaceutical composition comprising any one of the anti-CLL1 chimeric receptors described above, or any one of the engineered immune cells described above, and a pharmaceutically acceptable carrier. Further provided is a method of treating a disease (such as cancer) in an individual, comprising administering to the individual an effective amount of any one of the pharmaceutical compositions described above. In some embodiments, the engineered immune cell is autologous. In some embodiments, the engineered immune cell is allogenic. In some embodiments, the disease is cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the cancer is acute myeloid leukemia (AML). In some embodiments, the cancer is chronic myelogenous leukemia (CML). In some embodiments, the cancer is myelodysplastic syndromes (MDS). In some embodiments, wherein the immune cell expresses a safety-switch antigen or epitope, the method further comprises subsequently administering an effective amount of antibody that specifically binds to the safety-switch antigen or epitope.

[0031] One aspect of the present application provides a pharmaceutical composition comprising any one of the anti-CLL1 constructs described above and a pharmaceutically acceptable carrier. In some embodiments, there is provided a method of treating a disease (such as cancer) in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition. In some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of the anti-CLL1 construct according to any one of the anti-CLL1 constructs described above. In some embodiments, the cancer is acute myeloid leukemia (AML). In some embodiments, the cancer is chronic myelogenous leukemia (CML). In some embodiments, the cancer is myelodysplastic syndromes (MDS).

[0032] Also provided are methods of use, kits, and articles of manufacture comprising any one of the anti-CLL1 sdAbs, chimeric receptors, immune effector cell engagers, immunoconjugates, engineered immune cells, isolated nucleic acids, or vectors described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 depicts immune response of pre-immune serum and post-immune serum after final boost against human CLL1.

[0034] FIG. 2 depicts immune response of 4-chain antibodies (IgG1) and heavy-chain only antibodies (IgG2 and IgG3) in post-immune serum after boosts against human CLL1. Corresponding immunoglobulin fragments isolated from pre-immune serum were used as controls.

[0035] FIG. 3 depicts a schematic diagram of the chimeric antigen receptor (CAR) construct for screening.

[0036] FIG. 4 depicts results of a representative in vitro cytotoxicity assay showing cytolytic activity of CAR-T cells derived from various sdAbs against acute myeloid leukemia cell line THP-1. Un- transduced T cells (UnT) were used as the negative control, and CLL1 BM CAR-T cells were used as positive control.

[0037] FIGs. 5A-5D depict cytokine release in an in vitro cytotoxicity assay. FIG. 5A show levels of IFN-γ secreted by co-culture of anti-CLL1 CAR-T cells with THP-1 cells. FIG. 5B shows levels of IFN-γ secreted by anti-CLL1 CAR-T cells incubated alone. FIG. 5C shows levels of TNF-α secreted by co- culture of anti-CLL1 CAR-T cells with THP-1 cells. FIG. 5D shows levels of TNF-α secreted by anti- CLL1 CAR-T cells incubated alone.

[0038] FIGs. 6A-6C depict T cell populations in long-term co-cultures of anti-CLL1 CAR-T cells with THP-1 cells. FIG. 6A shows representative results by FACS analysis. FIG. 6B shows calculated T cell proliferation rates. FIG. 6C shows total T cell counts in the same co-cultures.

[0039] FIG. 7 depicts result of a representative in vivo efficacy assay showing anti-tumor ability of selected CAR-T cells in a U937-Luc xenograft mouse model. Un-transduced T cells ("UnT") were used as the negative control, and CLL1 BM CAR-T cells were used as positive control.

[0040] FIGs. 8A-8E depict schematic diagrams of CAR constructs, including regular CAR (FIG. 8A), Tandem CAR (FIG. 8B), Dual CARs (FIG. 8C), and Split CARs (FIGs. 8D-8E).

[0041] FIG. 8A shows an exemplary anti-CLL1 CAR comprising a CLL1 binding domain, a transmembrane domain, a CD28 or 4-1BB intracellular co-stimulatory sequence, and an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence.

[0042] FIG. 8B shows an exemplary tandem CAR comprising an extracellular domain comprising a CLL1 binding domain and a second antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a CD28 or 4-1BB intracellular co-stimulatory sequence and a CD3ζ intracellular signaling sequence.

[0043] FIG. 8C shows a dual CAR system comprising: (a) a first chimeric receptor comprising a CLL1 binding domain, a transmembrane domain, a CD28 or 4-1BB intracellular co-stimulatory sequence, and an intracellular signaling domain comprising a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence; and (b) a second chimeric receptor comprising a CD33 binding domain or an extracellular domain (ECD) of NKG2D ("NKG2D ECD"), a transmembrane domain, and an intracellular signaling domain comprising a CD28 or 4-1BB intracellular co-stimulatory sequence and a CD3ζ intracellular signaling sequence.

[0044] FIG. 8D shows a split CAR system comprising: (a) a first chimeric receptor comprising a CLL1 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a CD3\(\zeta\) intracellular signaling sequence; and (b) a second chimeric receptor comprising a CD33 binding domain or NKG2D ECD, a transmembrane domain, and an intracellular signaling domain comprising a CD28 or 4-1BB intracellular co-stimulatory sequence.

[0045] FIG. 8E shows a split CAR system comprising: (a) a first chimeric receptor comprising a CD33 binding domain (or a CD123 binding domain, or NKG2D ECD), a transmembrane domain, and an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence; and (b) a second chimeric receptor comprising a CLL1 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a CD28 or 4-1BB intracellular co-stimulatory sequence.

[0046] In FIGs. 8A-8E, the CLL1 binding domain may be any one of the anti-CLL1 sdAbs described herein. The second antigen binding domain may be NKG2D ECD or a CD33 binding domain (e.g., anti- CD33 sdAb), or a CD123 binding domain (e.g., anti-CD123 sdAb).

[0047] FIGs. 9A-9B depict results of a representative in vitro cytotoxicity assay showing cytolytic activity of AS82658-28z CAR-T cells against acute myeloid leukemia cell lines THP-1 (FIG. 9A) and MOLM-13 (FIG. 9B). Un-transduced T cells (UnT) were used as negative control, and CLL1 BM CAR-T cells were used as positive control.

[0048] FIGs. 10A-10B depict the potency of AS82658-28z CAR-T cells in inhibiting tumor cell growth in long-term co-cultures with U937 cells. FIG. 10A shows the cytotoxicity of CAR-T cells at various time points (3, 5, 7, 10, 12, 14, 17, 19, and 21 days after co-culture) as determined by FACS analysis. FIG. 10B shows T cell proliferation rates at various time points (3, 5, 7, 10, and 12 days after co-culture).

[0049] FIGs. 11A-11B depict cytokine release in long-term co-cultures assays. FIG. 11A shows levels of GM-CSF secreted by AS82658-28z CAR-T cells at various time points (3, 5, 7, 10, and 12 days after co-culture). FIG. 11B shows levels of IFN-y released by AS82658-28z CAR-T cells at various time points (3, 5, 7, 10, and 12 days after co-culture).

[0050] FIGs. 12A-12B depicts in vitro toxicity of AS82658-28z CAR-T cells against hematopoietic stem cells (HSC) as determined by CFU assays. UnT cells were used as negative control, and CLL1 BM CAR-T cells were used as positive control.

[0051] FIG. 13 depicts in vivo efficacy of AS82658-28z CAR-T cells in a U937-Luc xenograft mouse model. UnT cells were used as negative control, and CLL1 BM CAR-T cells were used as positive control.

[0052] FIG. 14 depicts in vitro cytotoxicity of CLL1 / CD33 tandem CAR-T cells against acute myeloid leukemia cell line THP-1. UnT cells were used as negative control, and AS82658-28z and AS49264 CAR-T cells (anti-CD33 CAR) were used as positive control.

[0053] FIG. 15A-15B depict the potency of CLL1 / CD33 tandem CAR-T cells in inhibiting tumor cell growth in long-term co-cultures with U937 cells. FIG. 15A shows cytotoxicity of CAR-T cells at various time points (2, 5, 7, 9, and 12 days after co-culture) as determined by FACS analysis. FIG. 15B shows T cell proliferation rates at various time points (2, 5, 7, and 9 days after co-culture).

[0054] FIGs. 16A-16B depict cytokine release in long-term co-cultures assays. FIG. 16A shows levels of GM-CSF secreted by CLL1 / CD33 tandem CAR-T cells at various time points (3, 6, and 9 days after co-culture). FIG. 16B shows levels of IFN-γ released by CLL1 / CD33 tandem CAR-T cells at various time points (2, 5, 7, and 9 days after co-culture).

[0055] FIGs. 17A-17C depict in vitro cytotoxicity of CLL1 / CD33 dual CAR-T cells against acute myeloid leukemia cell line THP-1. UnT cells were used as negative control.

[0056] FIG. 18A-18D depict in vivo efficacy of CLL1 / CD33 dual CAR-T and single-target CAR-T cells in HL-60-Luc xenograft model. FIG. 18A and 18C show the schematics of in vivo efficacy studies. FIG. 18B and 18D shows the curve of tumor growth in the HL-60-Luc xenograft mouse model after CAR-T cells treatment. UnT cells were used as negative control. DETAILED DESCRIPTION OF THE PRESENT APPLICATION

[0057] The present application provides anti-CLL1 single-domain antibodies (sdAbs) and constructs thereof, such as chimeric receptors, immune effector cell engagers, and immunoconjugates. Multivalent and multispecific chimeric receptors, dual chimeric receptor systems, and split chimeric receptor systems are also provided. The anti-CLL1 sdAbs, chimeric receptors, and engineered immune cells expressing the chimeric receptors described herein are useful agents for cancer treatment.

[0058] Accordingly, one aspect of the present application provides an anti-CLL1 construct comprising a single domain antibody ("sdAb") moiety that specifically binds to CLL1 (e.g., the extracellular domain of CLL1).

[0059] In another aspect, there is provided an anti-CLL1 chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (e.g., VHH), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular co-stimulatory sequence and / or a primary intracellular signaling sequence of an immune effector cell, e.g., a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence).

[0060] In another aspect, there is provided a multispecific chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (e.g., VHH) and a second antigen binding domain that specifically binds to a second antigen or epitope (e.g., an anti-CD33 sdAb, an anti-CD123 sdAb, or an extracellular domain of NKG2D), a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence and a primary intracellular signaling sequence of an immune effector cell (e.g., a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence).

[0061] In another aspect, there is provide a split chimeric receptor system comprising a first chimeric receptor comprising an anti-CLL1 sdAb (e.g., VHH), a transmembrane domain, and an intracellular signaling domain comprising a primary intracellular signaling sequence of an immune effector cell (e.g., a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence); and a second chimeric receptor comprising a second antigen binding domain that specifically binds to a second antigen or epitope (e.g., an anti-CD33 sdAb, an anti- CD123 sdAb, or an extracellular domain of NKG2D), a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence.

[0062] In another aspect, there is provide a split chimeric receptor system comprising a first chimeric receptor comprising an anti-CLL1 sdAb (e.g., VHH), a transmembrane domain, and an intracellular co- stimulatory sequence; and a second chimeric receptor comprising a second antigen binding domain that specifically binds to a second antigen or epitope (e.g., an anti-CD33 sdAb, an anti-CD123 sdAb, or an extracellular domain of NKG2D), a transmembrane domain, and an intracellular signaling domain comprising a primary intracellular signaling sequence of an immune effector cell (e.g., a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence).

[0063] Nucleic acids encoding the anti-CLL1 constructs, engineered immune cells (such as T cells) comprising the chimeric receptors or chimeric receptor systems, pharmaceutical compositions, kits, articles of manufacture and methods of treatment are also described herein. I. Definitions

[0064] The term "antigen" refers to any molecule capable of inducing an immune response in a host cell, or any molecule capable of binding to an antigen-specific receptor.

[0065] The term "antibody" or "antibody moiety" includes monoclonal antibodies (including full length 4-chain antibodies or full length heavy-chain only antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), as well as antibody fragments <semantics>(e.g.,Fab,F(ab′)2,and<annotation encoding="application / x-tex">(e.g., Fab, F(ab')_2, and< / annotation>< / semantics> Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. Antibodies contemplated herein include single-domain antibodies, such as heavy chain only antibodies.

[0066] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain <semantics>(VH)<annotation encoding="application / x-tex">(V_H)< / annotation>< / semantics> followed by three constant domains <semantics>(CH)<annotation encoding="application / x-tex">(C_H)< / annotation>< / semantics> for each of the <semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics> and <semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics> chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain <semantics>(VL)<annotation encoding="application / x-tex">(V_L)< / annotation>< / semantics> followed by a constant domain at its other end. The <semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics> is aligned with the <semantics>VH<annotation encoding="application / x-tex">V_H< / annotation>< / semantics> and the <semantics>CL<annotation encoding="application / x-tex">C_L< / annotation>< / semantics> is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated <semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>, <semantics>δ<annotation encoding="application / x-tex">\delta< / annotation>< / semantics>, <semantics>ϵ<annotation encoding="application / x-tex">\epsilon< / annotation>< / semantics>, <semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics> and <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>, respectively. The <semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics> and <semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics> classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

[0067] The term "heavy chain-only antibody" or "HCAb" refers to a functional antibody, which comprises heavy chains, but lacks the light chains usually found in 4-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0068] The term "single-domain antibody," "single-domain antibody moiety," "sdAb" or "sdAb moiety" refers to a single antigen-binding polypeptide having three complementary determining regions (CDRs), including full-length antibodies (e.g., HCAbs) and antigen-binding fragments thereof (e.g., VHH). The sdAb alone is capable of binding to the antigen without pairing with a corresponding CDR- containing polypeptide. In some cases, single-domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are referred herein as "VHHs". Some VHHs may also be known as Nanobodies. Camelid sdAb is one of the smallest known antigen-binding antibody fragments (see, e.g., Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). A basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.

[0069] An "isolated" antibody is one that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non- reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.

[0070] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as "VL" and "VL", respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. Heavy-chain only antibodies from the Camelid species have a single heavy chain variable region, which is referred to as "VHH". VHH is thus a special type of VH.

[0071] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody- dependent cellular toxicity.

[0072] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture or recombinantly, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application may be made by a variety of techniques, including, for example, the hybridoma method <semantics>(e.g.,<annotation encoding="application / x-tex">(e.g.,< / annotation>< / semantics> Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0073] The terms "full-length antibody," "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, full-length 4-chain antibodies include those with heavy and light chains including an Fc region. Full-length heavy-chain only antibodies include the heavy chain (such as VHH) and an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

[0074] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding and / or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ); single-chain antibody molecules; single-domain antibodies (such as VHH), and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produced two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0075] The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0076] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and - binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fy comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0077] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of the sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0078] "Functional fragments" of the antibodies described herein comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fc region of an antibody which retains or has modified FcR binding capability. Examples of antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0079] The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10) residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in greater detail in, for example, EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

[0080] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include PRIMATTZFD® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest. As used herein, "humanized antibody" is used a subset of "chimeric antibodies."

[0081] "Humanized" forms of non-human (e.g., camelid) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR (hereinafter defined) of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0082] A "human antibody" is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

[0083] The term "hypervariable region," "HVR," or "HV," when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, sdAbs comprise three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 displays the most diversity of the three HVRs, and is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0084] The term "Complementarity Determining Region" or "CDR" are used to refer to hypervariable regions as defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)

[0085] A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below in Table 1. Table 1. HVR delineations. [Image disponible dans le document PDF, Image available in the PDF document]

[0086] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0087] The amino acid residues of a sdAb (such as VHH) are numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195. According to this numbering, FR1 of a VHH comprises the amino acid residues at positions 1- 30, CDR1 of a VHH comprises the amino acid residues at positions 31-35, FR2 of a VHH comprises the amino acids at positions 36-49, CDR2 of a VHH comprises the amino acid residues at positions 50-65, FR3 of a VHH comprises the amino acid residues at positions 66-94, CDR3 of a VHH comprises the amino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113. In this respect, it should be noted that—as is well known in the art for VH domains and for VHH domains—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).

[0088] The expression "variable-domain residue-numbering as in Kabat" or "amino-acid-position numbering as in Kabat," and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

[0089] Unless indicated otherwise herein, the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra. The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0090] "Framework" or "FR" residues are those variable-domain residues other than the HVR residues as herein defined.

[0091] A "human consensus framework" or "acceptor human framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Examples include for the VL, the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al. Alternatively, a human consensus framework can be derived from the above in which particular residues, such as when a human framework residue is selected based on its homology to the donor framework by aligning the donor framework sequence with a collection of various human framework sequences. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0092] An "amino-acid modification" at a specified position, e.g. of the Fc region, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. Insertion "adjacent" to a specified residue means insertion within one to two residues thereof. The insertion may be N-terminal or C-terminal to the specified residue. The preferred amino acid modification herein is a substitution.

[0093] An "affinity-matured" antibody is one with one or more alterations in one or more HVRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration(s). In some embodiments, an affinity-matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation by VH- and VL -domain shuffling. Random mutagenesis of HVR and / or framework residues is described by, for example: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0094] As use herein, the term "specifically binds," "specifically recognizes," or is "specific for" refers to measurable and reproducible interactions such as binding between a target and an antigen binding protein (such as a chimeric receptor or an sdAb), which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antigen binding protein that specifically binds a target (which can be an epitope) is an antigen binding protein that binds this target with greater affinity, avidity, more readily, and / or with greater duration than it binds other targets. In some embodiments, the extent of binding of an antigen binding protein to an unrelated target is less than about 10% of the binding of the antigen binding protein to the target as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, an antigen binding protein that specifically binds a target has a dissociation constant (Kd) of <semantics>≤1μM<annotation encoding="application / x-tex">\leq 1 \mu M< / annotation>< / semantics>, <semantics>≤100 nM<annotation encoding="application / x-tex">\leq 100 \text{ nM}< / annotation>< / semantics>, <semantics>≤10 nM<annotation encoding="application / x-tex">\leq 10 \text{ nM}< / annotation>< / semantics>, or <semantics>≤0.1<annotation encoding="application / x-tex">\leq 0.1< / annotation>< / semantics> nM. In some embodiments, an antigen binding protein specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require exclusive binding.

[0095] The term "specificity" refers to selective recognition of an antigen binding protein (such as a chimeric receptor or an antibody construct) for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term "multispecific" as used herein denotes that an antigen binding protein has two or more antigen-binding sites of which at least two bind different antigens or epitopes. "Bispecific" as used herein denotes that an antigen binding protein has two different antigen-binding specificities. The term "monospecific" as used herein denotes an antigen binding protein that has one or more binding sites each of which bind the same antigen or epitope.

[0096] The term "valent" as used herein denotes the presence of a specified number of binding sites in an antigen binding protein. A natural antibody for example or a full length antibody has two binding sites and is bivalent. As such, the terms "trivalent", "tetravalent", "pentavalent" and "hexavalent" denote the presence of two binding site, three binding sites, four binding sites, five binding sites, and six binding sites, respectively, in an antigen binding protein.

[0097] "Antibody effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody—dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptors); and B cell activation. "Reduced or minimized" antibody effector function means that which is reduced by at least 50% (alternatively 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) from the wild type or unmodified antibody. The determination of antibody effector function is readily determinable and measurable by one of ordinary skill in the art. In a preferred embodiment, the antibody effector functions of complement binding, complement dependent cytotoxicity and antibody dependent cytotoxicity are affected. In some embodiments, effector function is eliminated through a mutation in the constant region that eliminated glycosylation, e.g., "effector-less mutation." In one aspect, the effector-less mutation is an N297A or DANA mutation (D265A+N297A) in the CH2 region. Shields et al., J. Biol. Chem. 276 (9): 6591-6604 (2001). Alternatively, additional mutations resulting in reduced or eliminated effector function include: K322A and L234A / L235A (LALA). Alternatively, effector function can be reduced or eliminated through production techniques, such as expression in host cells that do not glycosylate (e.g., E. coli.) or in which result in an altered glycosylation pattern that is ineffective or less effective at promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5): 3466-3473 (2003).

[0098] "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are required for killing of the target cell by this mechanism. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0099] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl- terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0100] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule <semantics>(e.g.,anantibodyoraCAR)<annotation encoding="application / x-tex">(e.g., an antibody or a CAR)< / annotation>< / semantics> and its binding partner <semantics>(e.g.,anantibodyoraCAR)<annotation encoding="application / x-tex">(e.g., an antibody or a CAR)< / annotation>< / semantics> antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen, or CAR and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present application. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

[0101] A "blocking" antibody or an "antagonist" antibody is one that inhibits or reduces a biological activity of the antigen it binds. In some embodiments, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0102] "Percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0103] "Chimeric receptor," "chimeric antigen receptor" or "CAR" as used herein refers to genetically engineered receptors, which can be used to graft one or more antigen specificity onto immune cells, such as T cells. Some chimeric receptors are also known as "artificial T-cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, the chimeric receptor comprises an extracellular domain specific for one or more antigens (such as tumor antigens) or epitopes, a transmembrane domain, and an intracellular signaling domain of a T cell and / or co-stimulatory receptors. "CAR-T" refers to a T cell that expresses a CAR. "Anti-CLL1 CAR" refers to a CAR having an extracellular binding domain specific for CLL1.

[0104] An "isolated" nucleic acid molecule encoding a chimeric receptor or an anti-CLL1 construct described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies herein existing naturally in cells.

[0105] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0106] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0107] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0108] As used herein, the term "autologous" is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0109] "Allogeneic" refers to a graft derived from a different individual of the same species.

[0110] The term "transfected" or "transformed" or "transduced" as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0111] As used herein, the expressions "cell", "cell line", and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transfectants" and "transfected cells" include the primary subject cell and cultures derived there from without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

[0112] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0113] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival. Also encompassed by "treatment" is a reduction of pathological consequence(s) of cancer. The methods of the present application contemplate any one or more of these aspects of treatment.

[0114] As used herein, an "individual" or a "subject" refers to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0115] The term "effective amount" used herein refers to an amount of an agent, such as an anti-CLL1 construct, an engineered immune cell, or a pharmaceutical composition thereof, sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and / or delay one or more of its symptoms. In reference to cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and / or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

[0116] "Adjuvant setting" refers to a clinical setting in which an individual has had a history of cancer, and generally (but not necessarily) been responsive to therapy, which includes, but is not limited to, surgery (e.g., surgery resection), radiotherapy, and chemotherapy. However, because of their history of cancer, these individuals are considered at risk of development of the disease. Treatment or administration in the "adjuvant setting" refers to a subsequent mode of treatment. The degree of risk <semantics>(e.g.,<annotation encoding="application / x-tex">(e.g.,< / annotation>< / semantics> when an individual in the adjuvant setting is considered as "high risk" or "low risk") depends upon several factors, most usually the extent of disease when first treated.

[0117] "Neoadjuvant setting" refers to a clinical setting in which the method is carried out before the primary / definitive therapy.

[0118] As used herein, "delaying" the development of cancer means to defer, hinder, slow, retard, stabilize, and / or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method that "delays" development of cancer is a method that reduces probability of disease development in a given time frame and / or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of individuals. Cancer development can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT Scan), Magnetic Resonance Imaging (MRI), abdominal ultrasound, clotting tests, arteriography, or biopsy. Development may also refer to cancer progression that may be initially undetectable and includes occurrence, recurrence, and onset.

[0119] The term "pharmaceutical formulation" refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. A "sterile" formulation is aseptic or free from all living microorganisms and their spores.

[0120] It is understood that embodiments of the present application described herein include "consisting" and / or "consisting essentially of" embodiments.

[0121] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0122] As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

[0123] The term "about X-Y" used herein has the same meaning as "about X to about Y."

[0124] As used herein and in the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. II. Anti-CLL1 Constructs

[0125] In one aspect, the present application provides an anti-CLL1 constructs comprising an anti- CLL1 sdAb moiety. Any one of the anti-CLL1 sdAbs described herein or antigen-binding fragments thereof (e.g., VHH) may be used in the anti-CLL1 construct. Anti-CLL1 sdAbs are described in Section "A. Anti-CLL1 single-domain antibodies" below.

[0126] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 94, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 94.

[0127] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 95, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 95.

[0128] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 96, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 96.

[0129] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 97, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 97.

[0130] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 98, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 98.

[0131] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb a sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 99, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 99.

[0132] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 100, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 100.

[0133] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 57, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 101, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 101.

[0134] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 60, a CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 64, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 102, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 102.

[0135] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 103, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 103.

[0136] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 74, a CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 78, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 104, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 104.

[0137] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 81, a CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 85, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 105, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 105.

[0138] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a CDR2 comprising the amino acid sequence of SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 92, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 106, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 106.

[0139] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 151, a CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 155, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 171, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 174.

[0140] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 158, a CDR2 comprising the amino acid sequence of SEQ ID NO: 160, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 162, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 172, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 175.

[0141] In some embodiments, there is provided an anti-CLL1 construct comprising an sdAb moiety that specifically binds to CLL1, wherein the sdAb moiety comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 165, a CDR2 comprising the amino acid sequence of SEQ ID NO: 167, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 169, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the sdAb moiety comprises the amino acid sequence of SEQ ID NO: 173, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SED ID NO: 176.

[0142] In some embodiments, the anti-CLL1 construct is a transmembrane molecule. In some embodiments, the anti-CLL1 construct is a secreted molecule.

[0143] In some embodiments, the anti-CLL1 construct is a monoclonal antibody comprising any one of the anti-CLL1 sdAbs described herein, including a camelid, chimeric, humanized or human antibody. In some embodiments, the anti-CLL1 construct is an antibody fragment, e.g., a VHH fragment. In some embodiments, the anti-CLL1 construct is a full-length heavy-chain only antibody comprising an Fc region of any antibody class or isotype, such as IgG1 or IgG4. In some embodiments, the Fc region has reduced or minimized effector function.

[0144] In some embodiments, the anti-CLL1 construct is a chimeric receptor comprising an extracellular domain comprising any one of the anti-CLL1 sdAbs described herein, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling sequence of an immune cell (e.g., a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence). In some embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence. In some embodiments, the intracellular signaling domain comprises both a primary intracellular signaling sequence of an immune cell (e.g., a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence) and an intracellular co-stimulatory sequence. Anti-CLL1 chimeric receptors and chimeric receptor systems are further described in Section "B. Chimeric receptors". Engineered immune cells comprising the anti-CLL1 chimeric receptors or chimeric receptor systems are described in Section IV.

[0145] In some embodiments, the anti-CLL1 construct is a monospecific molecule. In some embodiments, the anti-CLL1 construct is a multispecific molecule. In some embodiments, the anti-CLL1 construct is a bispecific molecule.

[0146] In some embodiments, the anti-CLL1 construct is a multispecific antigen binding protein comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein) and a second binding moiety that specifically binds to a second antigen or epitope. In some embodiments, the second binding moiety is an sdAb or an scFv. In some embodiments, the second binding moiety specifically binds to a different epitope on CLL1. In some embodiments, the second binding moiety specifically binds to a second antigen, such as a tumor antigen, or an antigen on the surface of an immune cell. In some embodiments, the anti-CLL1 sdAb is linked to the second binding moiety via a peptide linker.

[0147] In some embodiments, the anti-CLL1 construct is an immune effector cell engager comprising any one of the anti-CLL1 sdAbs described herein and a second binding moiety that specifically binds to an antigen on the surface of an immune cell, such as T cell. In some embodiments, the second binding moiety specifically binds to an antigen selected from the group consisting of CD3γ, CD3δ, CD3ε, CD3ζ, CD28, OX40, GITR, 4-1BB, CD27, CD40L, and HVEM. Immune effector cell engagers are further described in Section "C. Immune effector cell engagers" below.

[0148] In some embodiments, the anti-CLL1 construct is an immunoconjugate comprising any one of the anti-CLL1 sdAbs described herein and an effector molecule. In some embodiments, the effector molecule is a therapeutic agent selected from the group consisting of a drug, a toxin, a radioisotope, a protein, a peptide, and a nucleic acid. In some embodiments, the effector molecule is a drug or a toxin. In some embodiments, the effector molecule is a label. Immunoconjugates are further described in Section "D. Immunoconjugates" below. A. Anti-CLL1 single-domain antibodies

[0149] One aspect of the present application provides isolated single-domain antibodies (referred herein as "anti-CLL1 sdAbs") that specifically bind to CLL1. In some embodiments, the anti-CLL1 sdAb modulates CLL1 activity. In some embodiments, the anti-CLL1 sdAb is an antagonist antibody. Further provided are antigen-binding fragments (e.g., VHH) derived from any one of the anti-CLL1 sdAbs described herein, and constructs comprising any one of the anti-CLL1 sdAbs described herein. Exemplary anti-CLL1 sdAbs are listed in Table 2 below. The anti-CLL1 constructs described herein comprise one or more anti-CLL1 sdAb moieties. Table 2. Exemplary anti-CLL1 sdAbs. [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document] SEQ ID NO: 94 (AS82472 sdAb amino acid sequence; CDRs are underlined) QVQLVESGGDLVRPGGSLRLSCAASGFTFSIYDMNWVRQAPGKGLEWVAGISGNGYSTSYAESVKGRFTIS RDNAKNTVYLQLSSLKFEDTAMYYCVRDAERWDENDLRRKGQGTQVTVSS SEQ ID NO: 95 (AS82480 sdAb amino acid sequence; CDRs are underlined) EVQLVESGGGSVQAGGSLRLSCAASGVTYSSACMGWFRQAPGKGREVVAVLYAGGSTTHYASSVKERFTI SQDNAKNTVYLQMNSLKPEDTAVYYCAAALGDRSSCEWRYWGQGTQVTVSS SEQ ID NO: 96 (AS82494 sdAb amino acid sequence; CDRs are underlined) QVQLVESGGGLVQPGGSLRLSCAASGFTFSVYDMNWFRQAPGKGLEWVSGITGNGYTTSYADSVKGRFTI SRDNAKNTLYLQLNSLKSEDTAMYYCAKETNRGQGTQVTVSS SEQ ID NO: 97 (AS82505 sdAb amino acid sequence; CDRs are underlined) QVQLAESGGGLVQPGGSLRLSCVASGFTFSSYDMSWVRQAPGKGVEWVSTINSGGGSTYYAESAKGRFTI SRDNAKNTLYLQLNSLKTEDTAMYYCVKGFPDDDGPGELSREYNYWGQGTQVTVSS SEQ ID NO: 98 (AS82544 sdAb amino acid sequence; CDRs are underlined) EVQLVESGGALVQPGGSLRLSCTASGFLFRVYDMNWVRQAPGKGVEWIVGITNNGYTTAYADSVKGRFTI SRDNTENTLFLQMNSLKPEDTAMYYCQTDNGRVRGQGTQVTVSS SEQ ID NO: 99 (AS82658 sdAb amino acid sequence; CDRs are underlined) QVQLVESGGGSVQAGGALSLSCAASGYTVRIDYMGWYRQTPGKGREPVATIASNGGTAYADSVEGRFTIS QDNAKNSVYLQMNTLKPGDTAMYYCAAGTWPTLTYFGQGTQVTVSS SEQ ID NO: 100 (AS82718 sdAb amino acid sequence; CDRs are underlined) QVQLAESGGGLVQTGGSLRLSCTASGLNFGLYAMGWFRQAPGKEREGVSCINGGGGITVYSDFVKSRFTIS RDNAKNTLYLQMNSLKPDDTATYYCAADRSPFGSCSSDWSRSSDWSRMAEKFGYWGQGTQVTVSS SEQ ID NO: 101 (AS83180 sdAb amino acid sequence; CDRs are underlined) QVQLVESGGGSVQAGGSLRLSCVVSAATNCRYIAWYRQAPGKAREFVSTLGSDGNTNYADSVKGRFTISQ GNIKNMAYLEMNSLKPEDTGMYYCGTRCQIGDDWRSSDWAQGTQVTVSS SEQ ID NO: 102 (AS83183 sdAb amino acid sequence; CDRs are underlined) QVHLVESGGGSVQSGGSLRLSCAASGYAYRSYCMGWFRQAPGKVLEGVAAIESDGTTTYADSVMGRFTIS QDNAKNALYLQMNSLKPEDTAMYHCAAVKGSCDSASSDTPSYWGQGTQVTVSS SEQ ID NO: 103 (AS83309 sdAb amino acid sequence; CDRs are underlined) EVQLVESGGDLVRPGGSLRLSCAASGFTFSIYDMNWVRQAPGKGLEWVAGISGNGYSTSYAESVKGRFTIS KDNAKNTVYLQLSSLKFEDTAMYYCVRGGEKWDENDLRRKGQGTQVTVSS SEQ ID NO: 104 (AS83431 sdAb amino acid sequence; CDRs are underlined) QVRLVESGGGSVQSGGSLRLSCAASGYARSSTCLGWFRQAPGKEVEGVAIIGRDGSTGYADSVKGRFTISQ DNAKNTLYLHMDSLKPEDTAMYYCAAVEGGCEVSEGTGEQQLAYWGQGTQVTVSS SEQ ID NO: 105 (AS83478 sdAb amino acid sequence; CDRs are underlined) QVHLMESGGGLVQPGESLRLSCAASGFIFANYEMSWVRQAPGKVLEWVSGINSRGNATYYADSVKGRFTI SRDNAEHTLYLQMNSLKPEDTAMYHCVVGGMTTDQGSPDFYWGQGTQVTVSS SEQ ID NO: 106 (AS83591 sdAb amino acid sequence; CDRs are underlined) QVQLVESGGGLVQPGGSLRLSCAASGFLFSIYDMNWVRQAPGKGVEWIAGITNNEHTTAYADSVKGRFTIS RDNTKNTLFLQMNSLKPEDTAMYYCQRDDGQVRGQGTQVTVSS SEQ ID NO: 171 (AS83010 sdAb amino acid sequence; CDRs are underlined) QVQLVESGGGLVQPGGSLRLSCVASGFFFSAYDMNWFRQAPGKGLEWVSGITGNGYTTAYADSVKGRFTI SRDNAKNTLYLQLNSLKSEDTAMYYCTEGDNRGQGTQVTVSS SEQ ID NO: 172 (AS83457 sdAb amino acid sequence; CDRs are underlined) QVQLVESGGGLVQPGGSLRLSCAASGFFFSIYDMNWFRQAPGKGLEWVSGITGNGYTTAYADSVKGRFTIS RDNAKNTLYLQLNSLKSEDTAMYYCAQGSNRGRGTQVTVSS SEQ ID NO: 173 (AS83591 sdAb amino acid sequence; CDRs are underlined) QVQLVESGGGLVQPGGSLRLSCAASGFLFSIYDMNWVRQAPGKGVEWIAGITNNEHTTAYADSVKGRFTIS RDNTKNTLFLQMNSLKPEDTAMYYCQRDDGQVRGQGTQVTVSS SEQ ID NO: 107 (AS82472 sdAb nucleic acid sequence) CAGGTGCAGCTGGTGGAGTCTGGGGGGAGACTTGGTGCGGCCTGGGGGGGTCTCTGAGACTCTCCTGTGC AGCCTCTGGATTCACCTTCAGTATCTATGACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTCG AGTGGGTCGCAGGTATTAGTGGTAATGGTTACAGTACAAGCTATGCAGAGTCCGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGCCAAGAACACAGTGTATCTACAATTGAGCAGCCTGAAATTTGAAGACAC GGCCATGTATTACTGTGTAAGAGATGCGGAGAGGTGGGACGAGAATGACCTGCGACGGAAGGGCCAG GGGACCCAGGTCACCGTCTCCTCA SEQ ID NO: 108 (AS82480 sdAb nucleic acid sequence) GAGGTGCAACTGGTGGAGTCTGGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGC AGCCTCTGGCGTCACGTACAGTAGTGCCTGCATGGGCTGGTTCCGCCAGGCTCCAGGAAAGGGGCGCG AGGTGGTCGCGGTTCTTTATGCAGGTGGTAGTACCACACACTATGCCAGCTCCGTGAAGGAGCGATTC ACCATCTCCCAAGACAACGCCAAGAACACGGTATATCTGCAGATGAACAGCCTGAAACCTGAGGACA CTGCCGTTTACTACTGTGCGGCAGCTTTGGGTGATCGTTCAAGTTGCGAGTGGAGATACTGGGGCCAG GGGACCCAGGTCACCGTCTCCTCA SEQ ID NO: 109 (AS82494 sdAb nucleic acid sequence) CAGGTTCAGCTGGGGGGGGGGGGGGGGGGGGGGGGGGGG AGCCTCTGGATTCACCTTCAGTGTGTATGACATGAACTGGTTCCGCCAGGCTCCAGGGAAGGGACTCG AGTGGGTCTCAGGTATTACTGGGAATGGTTATACAACATCCTACGCAGACTCCGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAATTGAACAGCCTGAAAAGTGAGGACA CGGCCATGTATTACTGTGCAAAGGAGACTAATAGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA SEQ ID NO: 110 (AS82505 sdAb nucleic acid sequence) CAGGTGCAGCTGGCGGAGTCTGGGGGGGGGGCTTGGTGCAGCCTGGGGGGGTCTCTGAGACTCTCCTGTGT AGCCTCTGGATTCACCTTCAGTAGCTATGACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGAGTCG AGTGGGTCTCAACTATTAATAGTGGTGGTGGTAGTACATACTATGCAGAGTCCGCGAAGGGCCGATTT ACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAATTGAACAGCCTGAAAAACTGAGGACA CGGCCATGTATTACTGTGTAAAAAGGGTTTCCGGACGACGATGGACCGGGGGGAGTTAAGTAGAGAGTAT AATTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA SEQ ID NO: 111 (AS82544 sdAb nucleic acid sequence) GAGGTGCAGCTGGTGGAGTCTGGGGGGGGGGCCTTGGTGCAGCCTGGGGGGGTCTCTGAGACTCTCCTGTAC AGCCTCTGGATTTTTATTCCGTGTGTACGACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGCGTCG AGTGGATTGTAGGTATCACAAATAATGGTTATACCACAGCCTATGCAGACTCCGTGAAGGGCCGATTC ACCATCTCCAGAGACAACACCGAAAACACCCTGTTTCTGCAAATGAACAGCCTGAAACCTGAGGACAC GGCCATGTATTACTGTCAGACAGATAACGGTCGTGTGCGGGGCCAGGGGACCCAGGTCACCGTCTCCT CA SEQ ID NO: 112 (AS82658 sdAb nucleic acid sequence) CAGGTTCAGCTGGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGCTCTGAGCCTCTCCTGCGC AGCCTCTGGATACACCGTCAGAATCGACTACATGGGCTGGTACCGCCAGACTCCAGGGAAGGGCCGC GAGCCGGTCGCAACTATTGCCTCTAATGGTGGAACAGCCTATGCCGACTCCGTGGAGGGCCGATTTAC CATCTCCCAAGACAACGCCAAGAACTCGGTGTATCTGCAAATGAATACCCTGAAACCTGGGGACACTG CCATGTACTACTGTGCGGCGGGTACCTGGCCTACCTTGACTTACTT GTCTCCTCA SEQ ID NO: 113 (AS82718 sdAb nucleic acid sequence) CGGTGCAGCTGGTGGAATCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGTA GTCTCTGCAGCCACCAACTGTAGATACATTGCCTGGTACCGCCAGGCTCCAGGGAAGGCCCGCGAGTT CGTCTCAACTCTTGGTAGTGATGGTAACACAAACTACGCAGACTCCGTGAAGGGCCGATTCACTATCT CCCAAGGTAATATCAAGAACATGGCGTATCTGGAGATGAACAGCCTGAAACCTGAGGACACGGGCAT GTACTACTGCGGCACAAGGTGTCAAATTGGGGATGACTGGCGATCGAGCGACTGGGCCCAGGGGTGA AACCTGACGACACGCCACGTATTACTGTGCGGCAGACAGA TGGTCGCGCTCAAGCGATTGGTCGCGAATGGCGGAGAAGTTTGGTTATTGGGGCCCAGGGGACCCAGGT CACCGTCTCCTCA SEQ ID NO: 114 (AS83180 sdAb nucleic acid sequence) CAGGTGCAGCTGGTAGAATCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGT AGTCTCTGCAGCCACCAACTGTAGATACATTGCCTGGTACCGCCAGGCTCCAGGGAAGGCCCGCGAGT TCGTCTCAACTCTTGGTAGTGATGGTAACACAAACTACGCAGACTCCGTGAAGGGCCGATTCACTATC TCCCAAGGTAATATCAAGAACATGGCGTATCTGGAGATGAACAGCCTGAAACCTGAGGACACGGGCA TGTACTACTGCGGCACAAGGTGTCAAATTGGGGATGACTGGCGATCGAGCGACTGGGCCCAGGGGAC CCAGGTCACCGTCTCCTCA SEQ ID NO: 115 (AS83183 sdAb nucleic acid sequence) CAGGTGCACCTGGTGGAGTCTGGGGGGAGGCTCGGTGCAGTCTGGAGGGTCTCTGAGACTCTCCTGTGC AGCCTCTGGATACGCCTACCGTAGCTACTGTATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGTCCTCG AGGGGGTCGCAGCTATTGAGAGTGATGGTACTACAACCTACGCAGACTCCGTGATGGGCCGATTCACC ATCTCCCAAGACAACGCCAAGAATGCGCTCTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGG CCATGTATCACTGTGCGGCTGTCAAAGGGTCGTGCGATTCAGCGTCTTCCGACACCCCTAGTTACTGGG GCCAGGGACCCAGGTCACCGTCTCCTCA SEQ ID NO: 116 (AS83309 sdAb nucleic acid sequence) GAGGTGCAACTGGTGGAGTCTGGGGGGAGACTTGGTGCGGCCTGGGGGGGTCTCTGAGACTCTCCTGTGC AGCCTCTGGATTCACCTTCAGTATTTATGACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTCG AGTGGGTCGCAGGTATTAGTGGTAATGGTTACAGTACAAGCTATGCAGAGTCCGTGAAGGGCCGATTC ACCATCTCCAAAGACAACGCCAAGAACACAGTGTATCTACAATTGAGCAGCCTGAAATTTGAAGACAC GGCCATGTATTACTGTGTAAGAGGTGGGGAGAAGTGGGACGAAAATGACCTGCGACGGAAGGGCCAG GGGACCCAGGTCACCGTCTCCTCA SEQ ID NO: 117 (AS83431 sdAb nucleic acid sequence) CAGGTGAGGTTAGTGGAGTCTGGGGGGAGGCTCGGTGCAGTCTGGAGGGTCTCTGAGACTCTCCTGTGC AGCCTCTGGATATGCCCGCAGTAGTACTTGTTTGGGATGGTTCCGCCAGGCTCCAGGGAAGGAGGTCG AGGGGGTCGCAATTATTGGTAGGGATGGCAGTACGGGGTATGCAGACTCCGTGAAGGGCCGATTCAC CATCTCCCAAGACAACGCCAAGAACACGCTGTATCTACATATGGACAGCCTGAAACCTGAGGACACG GCTATGTATTACTGTGCGGCAGTTGAGGGCGGTTGTGAGGTGTCAGAAGGTACGGGGGAACAGCAGCT TGCTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA SEQ ID NO: 118(AS83478 sdAb nucleic acid sequence) CAGGTGCACCTGATGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGAGTCTCTGAGACTCTCCTGTGC CGCCTCTGGATTCATATTCGCTAACTACGAGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGTGCTCG AGTGGGTCTCAGGAATTAATAGCAGAGGTAATGCGACATACTATGCAGACTCCGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGCCGAGCACACGCTGTACCTCCAAATGAACAGCCTGAAACCTGAGGACA CGGCCATGTATCACTGTGTGGTAGGGGGTATGACCACTGATCAGGGCTCGCCAGATTTCTACTGGGGC CAGGGGACCCAGGTCACCGTCTCCA SEQ ID NO: 119 (AS83791 sdAb nucleic acid sequence) CAGGTGAAGTTGGTGGAGTCTTGGGGGGGGGCTTGGTGCAGCCTGGGGGGGTCTCTGAGACTCTCCTGTGT AGCCTCTGGATTCGCATTCAGTAGTGCCGACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGGTCG AAGCGGTCTCAGTTATTAATCGTGATGGTGCGAGCACATACTATGCGGACTCCGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGCCAAGAGCACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACA CGGCCATGTATCACTGTGCGGTAGTCCCGGAAAACGAATATGAAAGTGGATCGTATAACTACTGGGGC CAGGGGACCCAGGTCACCGTCTCCA SEQ ID NO: 174 (AS83010 sdAb nucleic acid sequence) CAGGTGCAGCTGGTGGAGTCTGGGGGGGGGGGTCTCTGAGACTCTCCTGTGT AGCCTCTGGATTCTTCTTCAGTGCGTATGACATGAACTGGTTCCGCCAGGCTCCAGGGAAGGGACTCG AGTGGGTCTCAGGTATTACTGGGAATGGTTATACGACCGCCTACGCAGACTCCGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAATTGAACAGCCTGAAAAGTGAGGACA CGGCCATGTATTACTGTACAGAGGGAGATAATAGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA SEQ ID NO: 175 (AS83457 sdAb nucleic acid sequence) CAGGTTCAGCTGGTGGAGTCTGGGGGGGGGGGTCTCTGAGACTCTCCTGTGC AGCCTCTGGATTCTTTTCAGTATTTATGACATGAACTGGTTCCGCCAGGCTCCAGGGAAGGGACTCGA GTGGGTCTCAGGTATTACTGGGAATGGTTATACGACCGCCTACGCAGACTCCGTGAAGGGCCGATTCA CCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAATTGAACAGCCTGAAAAGTGAGGACAC GGCCATGTATTACTGTGCACAGGGATCTAATAGGGGCCGGGGGACCCAGGTCACCGTCTCCTCA SEQ ID NO: 176 (AS83591 sdAb nucleic acid sequence) CAGGTTCAGCTGGTGGAGTCTGGGGGGAGGCTTGGTGCAGCCTGGGGGGGTCTCTGAGACTCTCCTGTGC AGCCTCTGGATTTTATTCAGTATTTACGACATGAATTGGGTCCGCCAGGCTCCAGGGAAGGGCGTCG AGTGGATCGCAGGTATTACAAATAATGAGCATACCACAGCCTATGCAGACTCCGTGAAGGGCCGATTC ACCATCTCCAGAGACAACACCAAAAACACCCTGTTTCTGCAAATGAACAGCCTGAAACCTGAGGACAC GGCCATGTATTACTGTCAGAGAGATGACGGACAAGTGCGGGGCCAGGGGACCCAGGTCACCGTCTCCT CA

[0150] C-type lectin-like molecule-1 (CLL1), also known as CLEC12A, C-type lectin domain family 12 member A, DCAL-2, MICL, and CD371, is a type II transmembrane glycoprotein that functions as an inhibitory receptor. The expression of CLL1 is restricted in myeloid lineage cells, as well as in the majority of AML blasts. In particular, CLL1 is selectively present on leukemic stem cells in acute myeloid leukemia (AML), but absent in normal hematopoietic stem cells. CLL1 can be a suitable tumor antigen target for immunotherapeutic agents against AML. See, e.g., Wang J. et al. (2018) J. Hematol. Oncol., 11:7; Zhao X. et al., (2010), Haematologica, 95(1): 71-78; and Lu H. et al. (2014) Angew Chem. Int. Ed. Engl. 53(37): 9841-9845.

[0151] In some embodiments, the anti-CLL1 sdAb specifically binds to human CLL1. In some embodiments, the anti-CLL1 sdAb specifically binds to cynomolgus monkey CLL1. In some embodiments, the anti-CLL1 sdAb specifically binds to the extracellular domain of CLL1. In some embodiments, the anti-CLL1 sdAb specifically binds to the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the anti-CLL1 sdAb specifically recognizes an epitope within human CLL1. In some embodiments, the anti-CLL1 sdAb cross-reacts with CLL1 from species other than human. In some embodiments, the anti-CLL1 sdAb is completely specific for human CLL1 and does not exhibit species or other types of non-human cross-reactivity.

[0152] In some embodiments, the anti-CLL1 sdAb cross-reacts with at least one allelic variant of the CLL1 protein (or fragments thereof). In some embodiments, the allelic variant has up to about 30 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30) amino acid substitutions (such as a conservative substitution) when compared to the naturally occurring CLL1 (or fragments thereof). In some embodiments, the anti-CLL1 sdAb does not cross-react with any allelic variant of the CLL1 protein (or fragments thereof).

[0153] In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 94. In some embodiments, there is provided an anti- CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 95. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 96. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 97. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 98. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 99. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 100. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 101. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 102. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 103. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 104. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 105. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 106. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 171. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 172. In some embodiments, there is provided an anti-CLL1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 173.

[0154] In some embodiments, there is provided an anti-CLL1 sdAb comprising at least one, at least two, or all three CDRs selected from: (a) a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, 74, 81, 88, 151, 158 and 165; (b) a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 6, 13, 20, 27, 34, 41, 48, 55, 62, 69, 76, 83, 90, 153, 160, and 167; and (c) a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 155, 162, and 169.

[0155] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, 74, 81, 88, 151, 158 and 165; (b) a CDR2 having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 6, 13, 20, 27, 34, 41, 48, 55, 62, 69, 76, 83, 90, 153, 160, and 167; and (c) a CDR3 having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 155, 162, and 169. In some embodiments, a CDR having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CLL1 sdAb comprising that sequence retains the ability to bind to CLL1. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 having about any one of 1, 2, or 3 amino acid substitutions (e.g., conservative substitutions), insertions, or deletions to the amino acid sequence of any one of SEQ ID NOs: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, 74, 81, 88, 151, 158 and 165; (b) a CDR2 having about any one of 1, 2, or 3 amino acid substitutions (e.g., conservative substitutions), insertions, or deletions to the amino acid sequence of any one of SEQ ID NOs: 6, 13, 20, 27, 34, 41, 48, 55, 62, 69, 76, 83, 90, 153, 160, and 167; and (c) a CDR3 having about any one of 1, 2, or 3 amino acid substitutions (e.g., conservative substitutions), insertions, or deletions to the amino acid sequence of any one of SEQ ID NOs: 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 155, 162, and 169.

[0156] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti- CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 4, 6 and 8.

[0157] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 11, 13 and 15.

[0158] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 20; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 20; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 18, 20 and 22.

[0159] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 25; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 27; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 25; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 27; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 25, 27 and 29.

[0160] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 34; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 34; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 32, 34 and 36.

[0161] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 39; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 41; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 39; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 41; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 39, 41 and 43.

[0162] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 46; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 48; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 46; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 48; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 46, 48 and 50.

[0163] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 55, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 57, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 55; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 57, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 55; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 53, 55 and 57.

[0164] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 62, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 64, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 62; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 64, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 62; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 60, 62 and 64.

[0165] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 67, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 69, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 67; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 67; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 67, 69 and 71.

[0166] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 74, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 76, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 78, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 74; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 76; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 78, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 74; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 76; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 74, 76 and 78.

[0167] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: $1, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 83, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 85, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 81; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 83; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 85, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 81; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 83; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 85. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 81, 83 and 85.

[0168] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 88, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 90, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 92, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 88; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 90; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 92, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 88; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 90; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 88, 90 and 92.

[0169] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 151, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 153, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 155, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 151; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 153; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 155, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 151; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 153; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 155. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 151, 153 and 155.

[0170] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 158, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 162, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 158; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 160; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 162, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 158; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 160; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 162. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 158, 160 and 162.

[0171] In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 165, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 167, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR2; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 169, or a variant thereof comprising up to about 3 (e.g., 1, 2, or 3) amino acid substitutions in the CDR3. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 165; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 167; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 169, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the CDRs. In some embodiments, there is provided an anti-CLL1 sdAb comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 165; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 167; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 169. In some embodiments, there is provided a polypeptide comprising the amino acid sequences of SEQ ID NOs: 165, 167 and 169.

[0172] In some embodiments, the anti-CLL1 sdAb, including any of the embodiments described above (i.e., anti-CLL1 sdAb comprising specific CDR1, CDR2, and / or CDR3) comprises a VHH domain having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171- 173. In some embodiments, a VHH sequence having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CLL1 sdAb comprising that sequence retains the ability to bind to CLL1. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173. In some embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In some embodiments, the anti-CLL1 sdAb comprises the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173, optionally including post- translational modifications of that sequence.

[0173] In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 94. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 94. In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 95. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 96. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 97. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 98. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 99. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 100. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 100. In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 101. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 101. In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 102. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 102. In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 103. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 103. In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 104. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 104. In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 105. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, there is provided an isolated anti-CLL1 sdAb comprising the amino acid sequence SEQ ID NO: 106. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 106. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 171. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 172. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 173.

[0174] In some embodiments, functional epitopes can be mapped by combinatorial alanine scanning. In this process, a combinatorial alanine-scanning strategy can be used to identify amino acids in the CLL1 protein that are necessary for interaction with anti-CLL1 sdAbs. In some embodiments, the epitope is conformational and crystal structure of anti-CLL1 sdAb bound to CLL1 may be employed to identify the epitopes.

[0175] In some embodiments, the present application provides antibodies (e.g., sdAbs) which compete with any one of the anti-CLL1 sdAbs described herein for binding to CLL1. In some embodiments, the present application provides antibodies (e.g., sdAbs) which compete with any one of the anti-CLL1 sdAbs provided herein for binding to an epitope on the CLL1. In some embodiments, an anti-CLL1 antibody (e.g., sdAb) is provided that binds to the same epitope as an anti-CLL1 sdAb comprising the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173. In some embodiments, an anti-CLL1 antibody (e.g., sdAbs) is provided that specifically binds to CLL1 competitively with an anti-CLL1 sdAb comprising the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173.

[0176] In some embodiments, competition assays may be used to identify a monoclonal antibody that competes with an anti-CLL1 sdAb described herein for binding to CLL1. Competition assays can be used to determine whether two antibodies bind the same epitope by recognizing identical or sterically overlapping epitopes or one antibody competitively inhibits binding of another antibody to the antigen. In certain embodiments, such a competing antibody binds to the same epitope that is bound by an antibody described herein. Exemplary competition assays include, but are not limited to, routine assays such as those provided in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.). In some embodiments, two antibodies are said to bind to the same epitope if each blocks binding of the other by 50% or more. In some embodiments, the antibody that competes with an anti-CLL1 sdAb described herein is a camelid, chimeric, humanized or human antibody. In some embodiments, the present application provides an antibody that competes with a camelid, chimeric, humanized, or human anti-CLL1 sdAb as described herein. B. Chimeric receptors and chimeric receptor systems

[0177] One aspect of the present application provides a chimeric receptor comprising an extracellular domain comprising one or more anti-CLL1 sdAbs (e.g., VHHs), a transmembrane domain, and an intracellular signaling domain. Also provided is a chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (e.g., VHH), a transmembrane domain, and an intracellular signaling domain; and (b) a second chimeric receptor comprising an extracellular domain comprising a binding moiety that specifically binds to a second antigen or epitope. Any one of the anti-CLL1 sdAbs described in Section A can be used in the chimeric receptors or chimeric receptor systems described herein. Exemplary structures of chimeric receptors and chimeric receptor systems are shown in FIGs. 8A-8E.

[0178] In some embodiments, there is provided a chimeric receptor targeting CLL1 (also referred herein as "anti-CLL1 chimeric receptor" or "anti-CLL1 CAR") comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein); (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the anti-CLL1 sdAb comprises any one of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 57, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60, a CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 64, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO: 74, a CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 78, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO: 81, a CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 85, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a CDR2 comprising the amino acid sequence of SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 92, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO: 151, a CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 155, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO: 158, a CDR2 comprising the amino acid sequence of SEQ ID NO: 160, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 162, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; or (16) a CDR1 comprising the amino acid sequence of SEQ ID NO: 165, a CDR2 comprising the amino acid sequence of SEQ ID NO: 167, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 169, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the anti-CLL1 sdAb moiety comprises a VHH domain comprising the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173. In some embodiments, the anti- CLL1 sdAb moiety is camelid, chimeric, human, or humanized. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling sequence of an immune effector cell (such as T cell). In some embodiments, the primary intracellular signaling sequence is derived from CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the primary intracellular signaling sequence is derived from CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> (i.e., "a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence"). In some embodiments, the intracellular signaling domain comprises an intracellular co- stimulatory sequence. In some embodiments, the intracellular co-stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In some embodiments, the intracellular co-stimulatory sequence is derived from CD28 or 4-1BB. In some embodiments, the intracellular signaling domain comprises both a primary intracellular signaling sequence (e.g., a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence) and an intracellular co-stimulatory sequence. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling sequence but does not comprise an intracellular co-stimulatory sequence. In some embodiments, the intracellular signaling domain comprises an intracellular co- stimulatory sequence but does not comprise a primary intracellular signaling sequence. In some embodiments, the anti-CLL1 chimeric receptor further comprises a hinge domain (e.g., a CD8 hinge domain) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the anti-CLL1 chimeric receptor further comprises a signal peptide (such as a CD8 signal peptide). In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the anti-CLL1 sdAb moiety, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD3ζ intracellular signaling sequence. In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the anti-CLL1 sdAb moiety, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence. In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the anti-CLL1 sdAb moiety, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28.

[0179] In some embodiments, there is provided an anti-CLL1 chimeric receptor comprising a polypeptide having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 120-132, 177-179, 181 and 229-230. In some embodiments, there is provided an anti-CLL1 chimeric receptor comprising a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 120-132, 177-179, 181 and 229-230. Also provided is a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 120-132, 177-179, 181 and 229-230.

[0180] Exemplary anti-CLL1 chimeric receptors are shown in Table 3 below. Table 3. Exemplary anti-CLL1 Chimeric Receptors. [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document]

[0181] In some embodiments, there is provided a chimeric receptor targeting CLL1 comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the anti-CLL1 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 99, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the primary intracellular signaling sequence is derived from CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>. In some embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence. In some embodiments, the anti-CLL1 chimeric receptor further comprises a hinge domain (e.g., a CD8 hinge domain) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the anti-CLL1 chimeric receptor further comprises a signal peptide (such as a CD8 signal peptide). In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the anti-CLL1 sdAb moiety, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB and a CD35 intracellular signaling sequence. In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the anti-CLL1 sdAb moiety, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from CD28 and a CD3ζ intracellular signaling sequence. In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 125 or 181, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 125 or 181.

[0182] In some embodiments, there is provided a chimeric receptor targeting CLL1 comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the anti-CLL1 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 94, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the primary intracellular signaling sequence is derived from CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>. In some embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence. In some embodiments, the anti-CLL1 chimeric receptor further comprises a hinge domain (e.g., a CD8 hinge domain) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the anti-CLL1 chimeric receptor further comprises a signal peptide (such as a CD8 signal peptide). In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the anti-CLL1 sdAb moiety, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB and a CD3\(2\) intracellular signaling sequence. In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the anti-CLL1 sdAb moiety, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from CD28 and a CD3ζ intracellular signaling sequence. In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 120 or 229, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 120 or 229.

[0183] In some embodiments, there is provided a chimeric receptor targeting CLL1 comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the anti-CLL1 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 96, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the primary intracellular signaling sequence is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence. In some embodiments, the anti-CLL1 chimeric receptor further comprises a hinge domain (e.g., a CD8 hinge domain) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the anti-CLL1 chimeric receptor further comprises a signal peptide (such as a CD8 signal peptide). In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the anti-CLL1 sdAb moiety, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB and a CD35 intracellular signaling sequence. In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the anti-CLL1 sdAb moiety, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from CD28 and a CD3\zeta intracellular signaling sequence. In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 122 or 230, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 122 or 230. Multivalent chimeric receptors

[0184] The present application also provides multivalent anti-CLL1 chimeric receptors that have two or more (such as about any one of 2, 3, 4, 5, 6, or more) binding moieties that specifically bind to CLL1. In some embodiments, one or more of the binding moieties are antigen binding fragments. In some embodiments, one or more of the binding moieties comprise sdAbs. In some embodiments, one or more of the binding moieties are derived from camelid antibodies. In some embodiments, one or more of the binding moieties are derived from a four-chain antibody. In some embodiments, one or more of the binding moieties are scFvs. In some embodiments, one or more of the binding moieties are derived from human antibodies. In some embodiments, one or more of the binding moieties are extracellular domains of receptors, polypeptide ligands or other non-antibody polypeptides that specifically bind to CLL1. In some embodiments, the multivalent chimeric receptor is monospecific, i.e., the multivalent chimeric receptor only targets CLL1, and comprises two or more binding sites for CLL1. In some embodiments, the multivalent chimeric receptor is multispecific, i.e., the multivalent chimeric receptor targets more than one antigen or epitope. The binding moieties specific for the same antigen may bind to the same epitope of the antigen (i.e., "mono-epitope chimeric receptor") or bind to different epitopes (i.e., "multi-epitope chimeric receptor" such as bi-epitope chimeric receptor or tri-epitope chimeric receptor) of the antigen. The binding sites specific for the same antigen may comprise the same or different sdAbs.

[0185] In some embodiments, the present application provides a multivalent (such as bivalent, trivalent, or of higher number of valencies) chimeric receptor comprising: (a) an extracellular domain comprising a plurality (such as at least about any one of 2, 3, 4, 5, 6, or more) of binding moieties specifically binding to CLL1; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0186] In some embodiments, the present application provides a multivalent (such as bivalent, trivalent, or of higher number of valencies) chimeric receptor comprising: (a) an extracellular domain comprising a plurality (such as at least about any one of 2, 3, 4, 5, 6, or more) of anti-CLL1 sdAb moieties; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0187] In some embodiments, the present application provides a multivalent (such as bivalent, trivalent, or of higher number of valencies) chimeric receptor comprising: (a) an extracellular domain comprising an sdAb moiety specifically binding to a first epitope of CLL1, and a second binding moiety (e.g., sdAb or scFv) specifically binding to a second epitope of CLL1; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first epitope and the second epitope are different. In some embodiments, the first anti-CLL1 sdAb is located at the N-terminus of the second CLL1 binding moiety (e.g., the second anti-CLL1 sdAb). In some embodiments, the first anti-CLL1 sdAb is located at the C-terminus of the second CLL1 binding moiety (e.g., the second anti-CLL1 sdAb). In some embodiments, the multivalent chimeric receptor specifically binds to two different epitopes on CLL1. In some embodiments, the multivalent chimeric receptor specifically binds to three or more different epitopes on CLL1.

[0188] In some embodiments, the binding moieties, such as sdAbs (including the plurality of sdAbs, or the first sdAb and / or the second sdAb) are camelid, chimeric, human, or humanized. In some embodiments, the binding moieties or sdAbs are fused to each other via peptide bonds or peptide linkers. In some embodiments, each peptide linker is no more than about 50 (such as no more than about any one of 35, 25, 20, 15, 10, or 5) amino acids long. In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling sequence of an immune effector cell (such as T cell). In some embodiments, the primary intracellular signaling sequence is derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the primary intracellular signaling sequence is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence. In some embodiments, the intracellular co-stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In some embodiments, the intracellular co-stimulatory sequence is derived from CD28 or 4-1BB. In some embodiments, the multivalent chimeric receptor further comprises a hinge domain (such as a CD8 hinge domain) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the multivalent chimeric receptor further comprises a signal peptide (such as a CD8 signal peptide). In some embodiments, the anti-CLL1 chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the extracellular domain, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD3\(z\) intracellular signaling sequence. In some embodiments, the multivalent chimeric receptor is monospecific. In some embodiments, the multivalent chimeric receptor is multispecific, such as bispecific.

[0189] The multivalent chimeric receptors describe herein may be specially suitable for targeting multimeric antigens via synergistic binding by the different antigen binding sites, or for enhancing binding affinity or avidity to the antigen. Any of the anti-CLL1 sdAbs described herein may be used in the extracellular domain of the multivalent chimeric receptors described herein. Multispecific chimeric receptor

[0190] The present application further provides multispecific chimeric receptors targeting two or more (such as about any one of 2, 3, 4, 5, 6, or more) different antigens. In some embodiments, the multispecific chimeric receptor has one antigen binding site for each antigen. In some embodiments, the multispecific chimeric receptor has more than two binding sites for at least one antigen. Each antigen binding site may comprise an sdAb. In some embodiments, the multispecific chimeric receptor is a bispecific chimeric receptor. In some embodiments, the multispecific chimeric receptor is a trispecific chimeric receptor.

[0191] In some embodiments, there is provided a multispecific (such as bispecific) chimeric receptor comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein) and a second binding domain that specifically binds to a second antigen or epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0192] In some embodiments, there is provided a multispecific (such as bispecific) chimeric receptor comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti- CLL1 sdAbs described herein) and a second sdAb that specifically binds to a second antigen; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0193] In some embodiments, there is provided a multispecific (such as bispecific) chimeric receptor comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein) and an scFv that specifically binds to a second antigen; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0194] In some embodiments, there is provided a multispecific (such as bispecific) chimeric receptor comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein) and an extracellular domain of a receptor that specifically binds to a second antigen; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0195] In some embodiments, the second antigen is selected from the group consisting of NKG2D ligands, CD33, WT1, CS1, CD123, Folate Receptor β, FLT3R, B7H6, TIM3, MUC1, c-kit, CD44v6, Lewis-Y, CD99, CD27 and CD70. In some embodiments, the anti-CLL1 sdAb and / or the second binding moiety (including second sdAb, or scFv) is camelid, chimeric, human, or humanized. In some embodiments, the anti-CLL1 sdAb and the second binding moiety (including second sdAb, scFv, or an extracellular domain of a receptor) are fused to each other via a peptide bond or a peptide linker. In some embodiments, the peptide linker is no more than about 50 (such as no more than about any one of 35, 25, 20, 15, 10, or 5) amino acids long. In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling sequence of an immune effector cell (such as T cell). In some embodiments, the primary intracellular signaling sequence is derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the primary intracellular signaling sequence is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence. In some embodiments, the intracellular co-stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In some embodiments, the intracellular co-stimulatory sequence is derived from CD28 or 4-1BB. In some embodiments, the multispecific chimeric receptor further comprises a hinge domain (such as a CD8 hinge domain) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the multispecific chimeric receptor further comprises a signal peptide (such as a CD8 signal peptide). In some embodiments, the multispecific chimeric receptor comprises a polypeptide comprising from the N-terminus to the C- terminus: a CD8 signal peptide, the extracellular domain, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD3ζ intracellular signaling sequence.

[0196] In some embodiments, there is provided a multispecific (e.g., bispecific) chimeric receptor comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein) and an anti-CD33 sdAb or scFv; (b) a transmembrane domain, and (c) an intracellular domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence.

[0197] In some embodiments, there is provided a multispecific (e.g., bispecific) chimeric receptor comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb and an anti-CD33 sdAb; (b) a transmembrane domain (e.g., a CD28 transmembrane domain), and (c) an intracellular domain (e.g., an intracellular domain comprising an intracellular co-stimulatory sequence derived from CD28 and a CD3 intracellular signaling sequence), wherein the anti-CLL1 sdAb comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; and wherein the anti-CD33 sdAb comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 198, a CDR2 comprising the amino acid sequence of SEQ ID NO: 200, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 202, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the anti- CLL1 sdAb is at the N-terminus of the anti-CD33 sdAb. In some embodiments, the anti-CLL1 sdAb is at the C-terminus of the anti-CD33 sdAb. In some embodiments, the anti-CLL1 sdAb is fused to the anti- CD33 sdAb via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID Nos: 142-147 and 182-183. In some embodiments, the anti-CLL1 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 99, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the anti-CD33 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 225, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 225. In some embodiments, the multispecific chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the extracellular domain, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from CD28 and a CD3ζ intracellular signaling sequence.

[0198] In some embodiments, there is provided a multispecific (e.g., bispecific) chimeric receptor comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb and an anti-CD33 sdAb, wherein the C-terminus of the anti-CLL1 sdAb is fused to the N-terminus of the anti-CD33 sdAb via a peptide linker; (b) a transmembrane domain (e.g., a CD28 transmembrane domain), and (c) an intracellular domain (e.g., an intracellular domain comprising an intracellular co-stimulatory sequence derived from CD28 and a CD3ζ intracellular signaling sequence), wherein the anti-CLL1 sdAb comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43; and wherein the anti-CD33 sdAb comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 198, a CDR2 comprising the amino acid sequence of SEQ ID NO: 200, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 202. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 147. In some embodiemnts, the peptide linker comprises the amino acid sequence of SEQ ID NO: 143. In some embodiemnts, the peptide linker comprises the amino acid sequence of SEQ ID NO: 183. In some embodiments, the anti-CLL1 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 99, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the anti-CD33 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 225, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 225. In some embodiments, the multispecific chimeric receptor comprises a polypeptide comprising from the N-terminus to the C- terminus: a CD8 signal peptide, the extracellular domain, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from CD28 and a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence. In some embodiments, the multispecific chimeric receptor comprises an amino acid sequence having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 184. In some embodiments, the multispecific chimeric receptor comprises an amino acid sequence having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 185. In some embodiments, the multispecific chimeric receptor comprises an amino acid sequence having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 188.

[0199] In some embodiments, there is provided a multispecific (e.g., bispecific) chimeric receptor comprising a polypeptide having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 184-195. In some embodiments, there is provided a multispecific (e.g., bispecific) chimeric receptor comprising a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 184-195. Also provided is a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 184-195.

[0200] Exemplary anti-CLL1 tandem chimeric receptors are shown in Table 4 below. Table 4. Exemplary anti-CLL1 sdAb Tandem Chimeric Receptors. [Image disponible dans le document PDF, Image available in the PDF document]

[0201] Any suitable anti-CD33 sdAb or scFv may be used for the multispecific chimeric receptor targeting CLL1 and CD33 described herein. Exemplary anti-CD33 sdAbs have been described, for example, see, PCT / CN2018 / 104882. Sequences of exemplary anti-CD33 sdAbs are shown in Table 5 below. Table 5. Exemplary anti-CD33 sdAbs. [Image disponible dans le document PDF, Image available in the PDF document] SEQ ID NO: 225 (AS49264 sdAb amino acid sequence; CDRs are underlined) EVQLVESGGGSVQAGGSLRLSCAASGYTYSINCMGWFRQAPGKEREGVAVISTGGGRTDYRDSVKGRFTI SQDNAKNTVYLQMNSLKPEDTAMYYCAGKTTYPGYGCGLGRSAYNYWGQGTQVTVSS SEQ ID NO: 226 (AS49814 sdAb amino acid sequence; CDRs are underlined) QIQLVESGGGSVQAGGSLRLSCVASGYIGGHYYMGWFRQAPGKEREGVAAIDIDSDGRTRYAGSVQGRFTI SQDNAKNTLHLQMSSLKPEDTGMYYCAVGVGWVPARLTPQAVSYWGKGTLVTVSS SEQ ID NO: 227 (AS50073 sdAb amino acid sequence; CDRs are underlined) QVQLVESGGGLVQAGGSLRLSCTASGFTFDNYVMGWFRQAPGKEREGVSCIGWSGGSTYYADSVKGRFTI SRDNAKNTLYLQMNSLKPEDTAMYYCAADQGKCSLGSAGADDMDYWGRGTLVTVSS SEQ ID NO: 228 (AS67190 sdAb amino acid sequence; CDRs are underlined) QVQLVESGGGLVQAGGSLRLSCAASGNVFRFNIMGWYRQAPGNQRELVASIDDGGDRSYADSVEGRFTIS RENGKKIMYLQMNSLKPEDTAVYYCAAGLGTYLNGRVSMATNYWGQGTQVTVSS

[0202] In some embodiments, there is provided a multispecific (e.g., bispecific) chimeric receptor comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein) and an anti-CD123 sdAb or scFv; (b) a transmembrane domain, and (c) an intracellular domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence.

[0203] In some embodiments, there is provided a multispecific (e.g., bispecific) chimeric receptor comprising: (a) an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein) and an extracellular domain of NKG2D; (b) a transmembrane domain, and (c) an intracellular domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence. Chimeric receptor systems

[0204] The present application further provides chimeric receptor systems comprising two or more chimeric receptors, including dual chimeric receptor systems and split chimeric receptors.

[0205] In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), a transmembrane domain, and an intracellular signaling domain comprising a primary intracellular signaling sequence of an immune cell (e.g., T cell) and an intracellular co-stimulatory sequence; (b) a second chimeric receptor comprising an extracellular domain comprising a second binding moiety (e.g., sdAb, scFv, or an extracellular domain of a receptor) that specifically binds to a second antigen or epitope, a transmembrane domain, and an intracellular signaling domain comprising a primary intracellular signaling sequence of an immune cell (e.g., T cell) and an intracellular co-stimulatory sequence. In some embodiments, the second antigen is selected from the group consisting of NKG2D ligands, CD33, WT1, CS1, CD123, Folate Receptor β, FLT3R, B7H6, TIM3, MUC1, c-kit, CD44v6, Lewis-Y, CD99, CD27 and CD70. In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152 and PD1. In some embodiments, the primary intracellular signaling sequence is derived from CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>, FcR<semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>, FcR<semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the primary intracellular signaling sequence is derived from CD3ζ. In some embodiments, the intracellular co- stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In some embodiments, the intracellular co-stimulatory sequence is derived from CD28 or 4-1BB. In some embodiments, the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain (such as a CD8 hinge domain) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the first chimeric receptor and / or the second chimeric receptor further comprises a signal peptide (such as a CD8 signal peptide). In some embodiments, the first chimeric receptor and the second chimeric receptor each comprises a polypeptide comprising from the N- terminus to the C-terminus: a CD8 signal peptide, the extracellular domain, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD3ζ intracellular signaling sequence.

[0206] In some embodiments, there is provided a split chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAB (such as any one of the anti-CLL1 sdAbs described herein), a transmembrane domain, and an intracellular signaling domain comprising a primary intracellular signaling sequence of an immune cell (e.g., T cell), wherein optionally the intracellular signaling domain does not comprise an intracellular co-stimulatory sequence; (b) a second chimeric receptor comprising an extracellular domain comprising a second binding moiety (e.g., sdAb, scFv, or an extracellular domain of a receptor) that specifically binds to a second antigen or epitope, a transmembrane domain, and an intracellular signaling domain comprising an intracellular co- stimulatory sequence, wherein optionally the intracellular signaling domain does not comprise a primary intracellular signaling sequence. In some embodiments, the second antigen is selected from the group consisting of NKG2D ligands, CD33, WT1, CS1, CD123, Folate Receptor β, FLT3R, B7H6, TIM3, MUC1, c-kit, CD44v6, Lewis-Y, CD99, CD27 and CD70. In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152 and PD1. In some embodiments, the primary intracellular signaling sequence is derived from CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>, FcR<semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the primary intracellular signaling sequence is derived from CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>. In some embodiments, the intracellular co- stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In some embodiments, the intracellular co-stimulatory sequence is derived from CD28 or 4-1BB. In some embodiments, the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain (such as a CD8 hinge domain) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the first chimeric receptor and / or the second chimeric receptor further comprises a signal peptide (such as a CD8 signal peptide). In some embodiments, the first chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the anti-CLL1 sdAb, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence. In some embodiments, the second chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the second binding domain, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28.

[0207] In some embodiments, there is provided a split chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain comprising an intracellular co-stimulatory sequence, wherein optionally the intracellular signaling domain does not comprise a primary intracellular signaling sequence; (b) a second chimeric receptor comprising an extracellular domain comprising a second binding moiety (e.g., sdAb, scFv, or an extracellular domain of a receptor) that specifically binds to a second antigen or epitope, a transmembrane domain, and an intracellular signaling domain comprising a primary intracellular signaling sequence of an immune cell (e.g., T cell), wherein optionally the intracellular signaling domain does not comprise an intracellular co-stimulatory sequence. In some embodiments, the second antigen is selected from the group consisting of NKG2D ligands, CD33, WT1, CS1, CD123, Folate Receptor β, FLT3R, B7H6, TIM3, MUC1, c-kit, CD44v6, Lewis-Y, CD99, CD27 and CD70. In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152 and PD1. In some embodiments, the primary intracellular signaling sequence is derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the primary intracellular signaling sequence is derived from CD3ζ. In some embodiments, the intracellular co-stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In some embodiments, the intracellular co-stimulatory sequence is derived from CD28 or 4-1BB. In some embodiments, the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain (such as a CD8 hinge domain) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the first chimeric receptor and / or the second chimeric receptor further comprises a signal peptide (such as a CD8) signal peptide). In some embodiments, the first chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the anti-CLL1 sdAb, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co- stimulatory sequence derived from 4-1BB or CD28. In some embodiments, the second chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the second binding domain, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence.

[0208] In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD35 intracellular signaling sequence; and (b) a second chimeric receptor comprising an extracellular domain comprising an anti-CD33 sdAb or scFv, a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD3ζ intracellular signaling sequence). In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152 and PD1. In some embodiments, the primary intracellular signaling sequence is derived from CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>. In some embodiments, the intracellular co-stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, Ligands of CD83 and combinations thereof. In some embodiments, the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain (such as a CD8 hinge domain) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the first chimeric receptor and / or the second chimeric receptor further comprises a signal peptide (such as a CD8 signal peptide).

[0209] Any suitable anti-CD33 sdAb or scFv may be used for the dual chimeric receptor systems targeting CLL1 and CD33 described herein. Exemplary anti-CD33 sdAbs have been described, for example, see, PCT / CN2018 / 104882. Sequences of exemplary anti-CD33 sdAbs are shown in Table 5.

[0210] In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb, a transmembrane domain (e.g., a CD28 transmembrane domain), and an intracellular signaling domain (e.g., an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from CD28 and a CD3ζ intracellular signaling sequence), wherein the anti-CLL1 sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; or (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; and (b) a second chimeric receptor comprising an extracellular domain comprising an anti-CD33 sdAb, a transmembrane domain (e.g., CD8 transmembrane domain), and an intracellular signaling domain (e.g., an intracellular signaling domain comprising an intracellular co- stimulatory sequence derived from 4-1BB and a CD3ζ intracellular signaling sequence), wherein the anti- CD33 sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR2 comprising the amino acid sequence of SEQ ID NO: 207, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 209, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 212, a CDR2 comprising the amino acid sequence of SEQ ID NO: 214, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 216, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; or (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 219, a CDR2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the anti-CLL1 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 94, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the anti-CLL1 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 96, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the anti-CD33 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 226, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 226. In some embodiments, the anti-CD33 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 227, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 227. In some embodiments, the anti-CD33 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 228, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 228. In some embodiments, the first chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the extracellular domain, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from CD28 and a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence. In some embodiments, the second chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the extracellular domain, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB and a CD3ζ intracellular signaling sequence.

[0211] In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb, a transmembrane domain (e.g., a CD28 transmembrane domain), and an intracellular signaling domain (e.g., an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from CD28 and a CD35 intracellular signaling sequence), wherein the anti-CLL1 sdAb comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and (b) a second chimeric receptor comprising an extracellular domain comprising an anti-CD33 sdAb, a transmembrane domain (e.g., CD8) transmembrane domain), and an intracellular signaling domain (e.g., an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB and a CD3ζ intracellular signaling sequence), wherein the anti-CD33 sdAb comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR2 comprising the amino acid sequence of SEQ ID NO: 207, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 209. In some embodiments, the anti-CLL1 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 94, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the anti-CD33 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 226, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 226. In some embodiments, the first chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the extracellular domain, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular signaling domain comprising an intracellular co- stimulatory sequence derived from CD28 and a CD3ζ intracellular signaling sequence. In some embodiments, the second chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the extracellular domain, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB and a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence.

[0212] In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb, a transmembrane domain (e.g., a CD28 transmembrane domain), and an intracellular signaling domain (e.g., an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from CD28 and a CD35 intracellular signaling sequence), wherein the anti-CLL1 sdAb comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and (b) a second chimeric receptor comprising an extracellular domain comprising an anti-CD33 sdAb, a transmembrane domain (e.g., CD8) transmembrane domain), and an intracellular signaling domain (e.g., an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB and a CD3ζ intracellular signaling sequence), wherein the anti-CD33 sdAb comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 212, a CDR2 comprising the amino acid sequence of SEQ ID NO: 214, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 216. In some embodiments, the anti-CLL1 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 94, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the anti-CD33 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 227, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 227. In some embodiments, the first chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the extracellular domain, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular signaling domain comprising an intracellular co- stimulatory sequence derived from CD28 and a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence. In some embodiments, the second chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the extracellular domain, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB and a CD3ζ intracellular signaling sequence.

[0213] In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb, a transmembrane domain (e.g., a CD28 transmembrane domain), and an intracellular signaling domain (e.g., an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from CD28 and a CD3ζ intracellular signaling sequence), wherein the anti-CLL1 sdAb comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and (b) a second chimeric receptor comprising an extracellular domain comprising an anti-CD33 sdAb, a transmembrane domain (e.g., CD8) transmembrane domain), and an intracellular signaling domain (e.g., an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB and a CD3ζ intracellular signaling sequence), wherein the anti-CD33 sdAb comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 219, a CDR2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-CLL1 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 96, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the anti-CD33 sdAb moiety comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 228, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 228. In some embodiments, the first chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the extracellular domain, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular signaling domain comprising an intracellular co- stimulatory sequence derived from CD28 and a CD3ζ intracellular signaling sequence. In some embodiments, the second chimeric receptor comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD8 signal peptide, the extracellular domain, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB and a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence.

[0214] In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising a polypeptide having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 229 or 230; and (b) a second chimeric receptor comprising a polypeptide having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 231, 232 or 233. In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 229 or 230; and (b) a second chimeric receptor comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 231, 232 or 233. In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 229; and (b) a second chimeric receptor comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 231. In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 229; and (b) a second chimeric receptor comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 232. In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 230; and (b) a second chimeric receptor comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 233.

[0215] In some embodiments, there is provided a dual chimeric receptor construct comprising a first polypeptide comprising any one of the anti-CLL1 chimeric receptor described herein and a second polypeptide comprising any one of the anti-CD33 chimeric receptor described herein, wherein the first polypeptide and the second polypeptide are fused to each other via a self-cleaving peptide (e.g., P2A) peptide). In some embodiments, the dual chimeric receptor construct comprises an amino acid sequence having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 234. In some embodiments, the dual chimeric receptor construct comprises an amino acid sequence having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 235. In some embodiments, the dual chimeric receptor construct comprises an amino acid sequence having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 236. In some embodiments, the dual chimeric receptor construct comprises the amino acid sequence of SEQ ID NO: 234, 235 or 236. Further provided are nucleic acid(s) encoding any one of the dual chimeric receptor constructs described herein. Also provided is a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 234-236.

[0216] Exemplary anti-CLL1 dual chimeric receptor systems are shown in Table 6 below. Table 6. Exemplary anti-CLL1 sdAb Dual Chimeric Receptors. [Image disponible dans le document PDF, Image available in the PDF document]

[0217] In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD35 intracellular signaling sequence; and (b) a second chimeric receptor comprising an extracellular domain comprising an anti-CD123 sdAb or scFv, a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD35 intracellular signaling sequence.

[0218] In some embodiments, there is provided a dual chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD35 intracellular signaling sequence; and (b) a second chimeric receptor comprising an extracellular domain comprising an extracellular domain of NKG2D, a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28 and a CD35 intracellular signaling sequence.

[0219] In some embodiments, there is provided a split chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), a transmembrane domain, and an intracellular signaling domain comprising a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence; and (b) a second chimeric receptor comprising an extracellular domain comprising an anti-CD33 sdAb or scFv, a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28.

[0220] In some embodiments, there is provided a split chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), a transmembrane domain, and an intracellular signaling domain comprising a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence; and (b) a second chimeric receptor comprising an extracellular domain comprising an anti-CD123 sdAb or scFv, a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28.

[0221] In some embodiments, there is provided a split chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), a transmembrane domain, and an intracellular signaling domain comprising a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence; and (b) a second chimeric receptor comprising an extracellular domain comprising an extracellular domain of NKG2D, a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28.

[0222] In some embodiments, there is provided a split chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28; and (b) a second chimeric receptor comprising an extracellular domain comprising an anti-CD33 sdAb or scFv, a transmembrane domain, and an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence.

[0223] In some embodiments, there is provided a split chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28; and (b) a second chimeric receptor comprising an extracellular domain comprising an anti-CD123 sdAb or scFv, a transmembrane domain, and an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence.

[0224] In some embodiments, there is provided a split chimeric receptor system comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence derived from 4-1BB or CD28; and (b) a second chimeric receptor comprising an extracellular domain comprising an extracellular domain of NKG2D, a transmembrane domain, and an intracellular signaling domain comprising a CD3ζ intracellular signaling sequence. Extracellular domain

[0225] The extracellular domain of the chimeric receptors described herein comprises one or more (such as any one of 1, 2, 3, 4, 5, 6 or more) binding moieties, such as sdAbs. In some embodiments, the one or more binding moieties are antibodies or antigen-binding fragments thereof. In some embodiments, the one or more binding moieties are derived from four-chain antibodies. In some embodiments, the one or more binding moieties are derived from camelid antibodies. In some embodiments, the one or more binding moieties are derived from human antibodies. In some embodiments, the one or more binding moieties are non-antibody binding proteins, such as extracellular domains of receptors, polypeptide ligands or engineered proteins that bind to an antigen. The binding moieties can be fused to each other directly via peptide bonds, or via peptide linkers.

[0226] In some embodiments, the extracellular domain comprises a second binding moiety. The second binding moiety specifically binds to a cell surface molecule. The second binding moiety may be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a special disease state. The antigens targeted by the second binding moiety may be directly or indirectly involved in the diseases. In some embodiments, the antigen is a tumor antigen. In some embodiments, the tumor antigen is associated with an acute myeloid leukemia (AML). In some embodiments, the tumor antigen is associated with chronic myelogenous leukemia (CML). In some embodiments, the tumor antigen is associated with myelodysplastic syndromes (MDS).

[0227] Tumor antigens are proteins that are produced by tumor cells that can elicit an immune response, particularly T-cell mediated immune responses. The selection of the targeted antigen of the invention will depend on the particular type of cancer to be treated. Exemplary tumor antigens include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), \(\beta\)-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0228] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell, and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development, when the immune system is immature, and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells, but which are expressed at much higher levels on tumor cells.

[0229] Non-limiting examples of TSA or TAA antigens include the following: Differentiation antigens such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER2 / 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 large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm- 23HI, 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, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0230] The second binding moiety can be of any suitable format. In some embodiments, the second binding moiety is derived from an antibody, such as a four-chain antibody, or a single-domain antibody, such as heavy-chain only antibody. In some embodiments, the second binding moiety is an antibody fragment, such as a Fab, Fv, scFv, or VHH. In some embodiments, the second binding moiety is an antibody fragment that specifically binds to an antigen selected from the group consisting of NKG2D ligands, CD33, WT1, CS1, CD123, Folate Receptor β, FLT3R, B7H6, TIM3, MUC1, c-kit, CD44v6, Lewis-Y, CD99, CD27 and CD70.

[0231] In some embodiments, the second binding moiety is a CD33-binding domain. In some embodiments, the CD33-binding domain is an antibody fragment (e.g., an scFv or a VHH) of an anti-CD33 antibody. In some embodiments, the CD33-binding domain is an scFv derived from hP67.6. In some embodiments, the CD33-binding domain is an anti-CD33 sdAb.

[0232] CD33, also known as Siglec-3 (sialic acid binding Ig-like lectin 3), gp67, or p67, is a transmembrane receptor expressed on cells of myeloid lineage. CD33 is the target of gemtuzumab ozogamicin (MYLOTARG®), an antibody-drug conjugate, which has been approved for treatment of patients with acute myeloid leukemia.

[0233] In some embodiments, the second binding moiety is a CD123-binding domain. In some embodiments, the CD123-binding domain is an antibody fragment (e.g., an scFv or a VHH) of an anti- CD123 antibody. In some embodiments, the CD123-binding domain is a ligand of CD123, or an IL-3 domain. In some embodiments, the IL-3 domain is derived from human IL-3, such as full-length or a functional fragment of human IL-3.

[0234] IL-3 (interleukin-3) gene is mapped on chromosome 5, encoding a protein 152 amino acids long. IL-3 is a cytokine, capable of supporting a broad range of cellular activities such as cell growth, differentiation and apoptosis. IL-13 acts by binding to the interleukin-3 receptor (IL-3R), also known as CD123 antigen. IL-3R is a heterodimeric receptor, comprising a ligand specific alpha subunit and a signal transducing beta subunit, shared by the receptors for IL-3, colony stimulating factor 2 (CSF2 / GM-CSF), and interleukin 5 (IL5). Activation of the IL-3R results in the phosphorylation of the βc chain, recruitment of SH2-containing adaptor molecules such as Vav1, and downstream signal transduction via Jak2 / STAT5 and the Ras / MAPK pathway.

[0235] IL-3R is a 75 kD glycoprotein and becomes 43 kD when hydrolyzed by N-glycosidase. IL-3R has three extracellular domains which are responsible for specific binding to IL-3, a transmembrane domain, and a short intercellular domain which is indispensable for intracellular signaling (Sato et al. 1993). IL-3R is a heterodimeric receptor with low affinity and high specificity for IL-3. Upon binding to IL-3, the IL-3R is activated and promotes cell proliferation and survival (Liu et al. 2015).

[0236] CD123 is overexpressed on AML blasts (i.e., myelobasts). AML blasts and leukemia stem cells (LSCs) in 75 to 89% of AML patients express CD123. In sharp contrast, there is low or undetectable expression of CD123 on normal hematopoietic stem cells (HSCs) (Frankel et al. 2014; Jordan et al. 2000). Apart from AML, CD123 is also overexpressed in a variety of hematologic malignancies, including B cell lineage acute lymphoblastic leukemia, chronic myeloid leukemia, plasmacytoid dendritic cell neoplasm, and hairy cell leukemia (Munoz et al. 2001). This expression profile makes CD123 a valuable biomarker in clinical diagnosis, prognosis and intervention of the diseases. Currently, early phase clinical trials have demonstrated that CD123-targeting therapies are safe and without major adverse effects on hematopoiesis. The anti-leukemic activities of CD123-targeting therapies in humans are still being investigated.

[0237] In some embodiments, the second binding moiety is a ligand, or a ligand binding domain of a receptor, such as an extracellular domain of a receptor. In some embodiments, the second binding moiety is a ligand or ligand binding domain derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the second binding moiety is an extracellular domain of NKG2D. In some embodiments, the second binding moiety comprises the amino acid sequence of SEQ ID NO: 149.

[0238] SEQ ID NO: 149 NKG2D-L binding domain FNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLL KLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYIC MQRTV

[0239] NKG2D is a unique member of the NKG2 family, which are C-type lectin receptors that stimulate or inhibit cytotoxic activity of NK cells. NKG2D is a type II transmembrane-anchored glycoprotein, expressed primarily on the surface of NK cells and CD8+T cells (e.g., <semantics>αβ<annotation encoding="application / x-tex">\alpha\beta< / annotation>< / semantics> T cells and <semantics>γδ<annotation encoding="application / x-tex">\gamma\delta< / annotation>< / semantics> T cells). It is highly conserved across multiple species, with 70% sequence identity shared between the human and murine receptors. Unlike the other NKG2 receptors that heterodimerize with CD94 and bind to nonclassical MHC glycoproteins class I, NKG2D forms homodimers and bind to cellular stress-inducible molecules. Accumulating evidence indicates that NKG2D plays a crucial role in immunosurveillance against stressed or abnormal cells, such as autologous tumor cells and virus-infected cells.

[0240] A variety of NKG2D ligands have been identified in humans, including MIC molecules (MHC class I chain-related proteins A and B, or MICA and MICB) encoded by genes in the MHC family, and ULBP molecules (UL16-binding proteins, also known as RAET1 proteins) which are clustered on human chromosome 6 (Bahram et al. 2005). All NKG2D ligands are homologous to MHC class I molecules and exhibit considerable allelic variation. Although NKG2D ligand RNAs are broadly expressed on all tissues and organs of the body, NKG2D ligands are generally absent from the surface of normal adult cells (Le Bert and Gasser 2014). However, the expression of NKG2D ligands is induced or upregulated primarily in tissues of epithelial origin in response to cellular stress, including heat shock, DNA damage, and stalled DNA replication. Presence of NKG2D ligands on a cell flags the cell for NK cell targeting and potential elimination (Le Bert and Gasser 2014). Interestingly, high activity of DNA repair pathways in transformed cells across a variety of hematologic and solid tumors lead to expression of NKG2D ligands, which renders these cells susceptible to NK-mediated lysis (Sentman et al. 2006).

[0241] NKG2D is encoded by KLRK1 gene. NKG2D is a transmembrane receptor protein comprising three domains: cytoplasmic domain (residues 1-51 of human NKG2D), transmembrane domain (residues 52-72 of human NKG2D), and extracellular domain (residues 73-216 of human NKG2D). The extracellular domain of NKG2D contains a C-type lectin domain (residues 98-213 of human NKG2D). Transmembrane domain

[0242] The chimeric receptors of the present application comprise a transmembrane domain that can be directly or indirectly fused to the extracellular domain. The transmembrane domain may be derived either from a natural or from a synthetic source. As used herein, a "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. Transmembrane domains compatible for use in the chimeric receptors described herein may be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0243] Transmembrane domains are classified based on the three dimensional structure of the transmembrane domain. For example, transmembrane domains may form an alpha helix, a complex of more than one alpha helix, a beta-barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell. Furthermore, transmembrane domains may also or alternatively be classified based on the transmembrane domain topology, including the number of passes that the transmembrane domain makes across the membrane and the orientation of the protein. For example, single-pass membrane proteins cross the cell membrane once, and multi-pass membrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 or more times). Membrane proteins may be defined as Type I, Type II or Type III depending upon the topology of their termini and membrane-passing segment(s) relative to the inside and outside of the cell. Type I membrane proteins have a single membrane-spanning region and are oriented such that the N-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is present on the cytoplasmic side. Type II membrane proteins also have a single membrane-spanning region but are oriented such that the C-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the N-terminus of the protein is present on the cytoplasmic side. Type III membrane proteins have multiple membrane- spanning segments and may be further sub-classified based on the number of transmembrane segments and the location of N- and C-termini.

[0244] In some embodiments, the transmembrane domain of the CAR described herein is derived from a Type I single-pass membrane protein. In some embodiments, transmembrane domains from multi-pass membrane proteins may also be compatible for use in the CARs described herein. Multi-pass membrane proteins may comprise a complex (at least 2, 3, 4, 5, 6, 7 or more) alpha helices or a beta sheet structure. Preferably, the N-terminus and the C-terminus of a multi-pass membrane protein are present on opposing sides of the lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side.

[0245] In some embodiments, the transmembrane domain of the chimeric receptor comprises a transmembrane domain chosen from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CLL1, CD37, CD64, CD80, CD86, CD134, 4-1BB, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4- 1BB (4-1BB), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152 and PD1.

[0246] In some embodiments, the transmembrane domain is derived from CD8. In some embodiments, the transmembrane domain is a transmembrane domain of CD8α comprising the amino acid sequence of IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 133). In some embodiments, the transmembrane domain is derived from CD28. In some embodiments, the transmembrane domain is a transmembrane domain of CD28 comprising the amino acid sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 134).

[0247] Transmembrane domains for use in the chimeric receptors described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least approximately 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in U.S. Patent No.7,052,906 B1 and PCT Publication No. WO 2000 / 032776 A2.

[0248] The transmembrane domain may comprise a transmembrane region and a cytoplasmic region located at the C-terminal side of the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids and, in some embodiments, helps to orient the transmembrane domain in the lipid bilayer. In some embodiments, one or more cysteine residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

[0249] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of the chimeric receptor comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan and valine may be present at the C terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises mostly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence. The hydropathy, or hydrophobic or hydrophilic characteristics of a protein or protein segment, can be assessed by any method known in the art, for example the Kyte and Doolittle hydropathy analysis. Intracellular signaling domain

[0250] The chimeric receptors of the present application comprise an intracellular signaling domain. The intracellular signaling domain of a single chimeric receptor or the intracellular signaling domains of two chimeric receptors in a chimeric receptor system is responsible for activation of at least one of the normal effector functions of the immune effector cell expressing the chimeric receptor(s). The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term "cytoplasmic signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire cytoplasmic signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term cytoplasmic signaling domain is thus meant to include any truncated portion of the cytoplasmic signaling domain sufficient to transduce the effector function signal.

[0251] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling sequence of an immune effector cell. In some embodiments, the chimeric receptor comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling sequence of an immune effector cell. "Primary intracellular signaling sequence" refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. In some embodiments, the primary intracellular signaling sequence contains a signaling motif known as immunoreceptor tyrosine-based activation motif, or ITAM. An "ITAM," as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells. The motif may comprises two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. ITAMs may also function as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3ζ, FcR gamma(FCER1G), FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0252] In some embodiments, the primary intracellular signaling sequence is derived from CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>, i.e., the primary intracellular signaling sequence is a CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence. In some embodiments, the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>. In some embodiments, the primary intracellular signaling sequence is a cytoplasmic signaling domain of wildtype CD3ζ. In some embodiments, the primary intracellular signaling sequence is a functional mutant of the cytoplasmic signaling domain of CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> containing one or more mutations, such as Q65K. In some embodiments, the CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> intracellular signaling sequence comprises the amino acid sequence of SEQ ID NO: 135.

[0253] SEQ ID NO: 135 CD3ζ intracellular signaling sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0254] In some embodiments, the intracellular signaling domain comprises an intracellular co- stimulatory sequence. In some embodiments, the intracellular signaling domain consists of an intracellular co-stimulatory sequence. In some embodiments, the intracellular signaling domain does not comprise a primary intracellular signaling sequence of an immune effector cell (e.g., T cell). In some embodiments, the intracellular signaling domain comprises both a primary intracellular signaling sequence of an immune effector cell (e.g., T cell) and an intracellular co-stimulatory sequence. In some embodiments, the intracellular signaling domain does not comprise an intracellular co-stimulatory sequence. In some embodiments, the first chimeric receptor comprises an intracellular signaling domain comprising a primary intracellular signaling sequence of an immune effector cell (e.g., T cell), and the second chimeric receptor comprises an intracellular signaling domain comprising an intracellular co-stimulatory sequence. In some embodiments, the first chimeric receptor comprises an intracellular signaling domain comprising an intracellular co-stimulatory sequence, and the second chimeric receptor comprises an intracellular signaling domain comprising a primary intracellular signaling sequence of an immune effector cell <semantics>(e.g.,<annotation encoding="application / x-tex">(e.g.,< / annotation>< / semantics> T cell).

[0255] Many immune effector cells require co-stimulation, in addition to stimulation of an antigen- specific signal, to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell. In some embodiments, the chimeric receptor comprises at least one intracellular co- stimulatory sequence. The term "intracellular co-stimulatory sequence," as used herein, refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function. The intracellular co-stimulatory sequence of the chimeric receptor described herein can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. "Intracellular co-stimulatory sequence" can be the cytoplasmic portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to a cognate binding partner on an immune cell (such as T cell) that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival.

[0256] In some embodiments, the intracellular signaling domain comprises a single intracellular co- stimulatory sequence. In some embodiments, the intracellular signaling domain comprises two or more (such as about any of 2, 3, 4, or more) intracellular co-stimulatory sequences. In some embodiments, the intracellular signaling domain comprises two or more of the same intracellular co-stimulatory sequences, for example, two copies of the intracellular co-stimulatory sequence of CD28. In some embodiments, the intracellular signaling domain comprises two or more intracellular co-stimulatory sequences from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling sequence (such as cytoplasmic signaling domain of CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>) and one or more intracellular co-stimulatory sequences. In some embodiments, the one or more intracellular co-stimulatory sequences and the primary intracellular signaling sequence (such as cytoplasmic signaling domain of CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>) are fused to each other via optional peptide linkers. The primary intracellular signaling sequence, and the one or more intracellular co-stimulatory sequences may be arranged in any suitable order. In some embodiments, the one or more intracellular co-stimulatory sequences are located between the transmembrane domain and the primary intracellular signaling sequence (such as cytoplasmic signaling domain of CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>). Multiple intracellular co-stimulatory sequences may provide additive or synergistic stimulatory effects.

[0257] Activation of an intracellular co-stimulatory sequence in a host cell (e.g., an immune cell) may induce the cell to increase or decrease the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The intracellular co-stimulatory sequence of any co-stimulatory molecule may be compatible for use in the chimeric receptors described herein. The type(s) of intracellular co-stimulatory sequence is selected based on factors such as the type of the immune effector cells in which the effector molecules would be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect). Examples of intracellular co-stimulatory sequences for use in the chimeric receptors can be the cytoplasmic signaling domain of co-stimulatory proteins, including, without limitation, members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); members of the TNF superfamily (e.g.,4-1BB / TNFSF9 / 4-1BB, 4-1BB Ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 Ligand / TNFSF7, CD30 / TNFRSF8, CD30 Ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 Ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR Ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lymphotoxin-alpha / TNF-beta, OX40 / TNFRSF4, OX40 Ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-alpha, and TNF RII / TNFRSF1B); members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150); and any other co-stimulatory molecules, such as CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA Class I, HLA- DR, Ikaros, Integrin alpha 4 / CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-1), and NKG2C.

[0258] In some embodiments, the one or more intracellular co-stimulatory sequences are selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specially bind to CD83.

[0259] In some embodiments, the intracellular signaling domain in the chimeric receptor of the present application comprises an intracellular co-stimulatory sequence derived from CD28. In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> and an intracellular co-stimulatory sequence of CD28. In some embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence of CD28 comprising the amino acid sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP PRDFAAYRS (SEQ ID NO: 136).

[0260] In some embodiments, the intracellular signaling domain in the CAR of the present application comprises an intracellular co-stimulatory sequence derived from 4-1BB (i.e., CD137). In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3ζ and an intracellular co-stimulatory sequence of 4-1BB. In some embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence of 4-1BB comprising the amino acid sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCEL (SEQ ID NO: 137).

[0261] In some embodiments, the intracellular signaling domain in the CAR of the present application comprises an intracellular co-stimulatory sequence derived from ICOS. In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3<semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics> and an intracellular co- stimulatory sequence of ICOS. In some embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence of ICOS comprising the amino acid sequence of CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSR LTDVTL (SEQ ID NO: 138).

[0262] In some embodiments, the intracellular signaling domain in the CAR of the present application comprises an intracellular co-stimulatory sequence of CD28 and an intracellular co-stimulatory sequence of 4-1BB. In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3ζ, an intracellular co-stimulatory sequence of CD28, and an intracellular co-stimulatory sequence of 4-1BB.

[0263] In some embodiments, the intracellular signaling domain comprises a polypeptide comprising a cytoplasmic signaling domain of CD3ζ. In some embodiments, the intracellular signaling domain comprises a polypeptide comprising an intracellular co-stimulatory sequence of CD28. In some embodiments, the intracellular signaling domain comprises a polypeptide comprising an intracellular co- stimulatory sequence of 4-1BB. In some embodiments, the intracellular signaling domain comprises a polypeptide comprising from the N-terminus to the C-terminus: an intracellular co-stimulatory sequence of 4-1BB, and a cytoplasmic signaling domain of CD3ζ. In some embodiments, the intracellular signaling domain comprises a polypeptide comprising from the N-terminus to the C-terminus: an intracellular co-stimulatory sequence of CD28, an intracellular co-stimulatory sequence of 4-1BB, and a cytoplasmic signaling domain of CD3ζ.

[0264] Also within the scope of the present disclosure are variants of any of the intracellular co- stimulatory sequences described herein, such that the intracellular co-stimulatory sequence is capable of modulating the immune response of the immune cell. In some embodiments, the intracellular co- stimulatory sequences comprises up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) as compared to a wild-type counterpart. Such intracellular co-stimulatory sequences comprising one or more amino acid variations may be referred to as variants. Mutation of amino acid residues of the intracellular co-stimulatory sequence may result in an increase in signaling transduction and enhanced stimulation of immune responses relative to intracellular co-stimulatory sequences that do not comprise the mutation. Mutation of amino acid residues of the intracellular co-stimulatory sequence may result in a decrease in signaling transduction and reduced stimulation of immune responses relative to intracellular co- stimulatory sequences that do not comprise the mutation. Hinge region

[0265] The chimeric receptors of the present application may comprise a hinge domain that is located between the extracellular domain and the transmembrane domain. A hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular domain relative to the transmembrane domain of the effector molecule can be used.

[0266] The hinge domain may contain about 10-100 amino acids, e.g., about any one of 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. In some embodiments, the hinge domain may be at least about any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length. |0267| In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8, such as CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. In some embodiments, the hinge domain of CD8\alpha comprises the amino acid sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 139). In some embodiments, the hinge domain is derived from CD28. In some embodiments, the hinge domain of CD28 comprises the amino acid sequence of IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 140).

[0268] Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the chimeric receptor systems described herein. In some embodiments, the hinge domain is the hinge domain that joins the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.

[0269] Non-naturally occurring peptides may also be used as hinge domains for the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain of an Fc receptor and the N- terminus of the transmembrane domain is a peptide linker, such as a (GxS)n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more. In some embodiments, the peptide linker comprises the amino acid sequence of GGGGSGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG Signal peptide

[0270] The chimeric receptors of the present application may comprise a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. In general, signal peptides are peptide sequences that target a polypeptide to the desired site in a cell. In some embodiments, the signal peptide targets the effector molecule to the secretory pathway of the cell and will allow for integration and anchoring of the effector molecule into the lipid bilayer. Signal peptides including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences may be compatible for use in the chimeric receptors described herein. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8, GM-CSF receptor α, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8, such as CD8α. In some embodiments, the signal peptide of CD8α comprises the amino acid sequence of MALPVTALLLPLALLLHAARP (SEQ ID NO: 142). C. Immune effector cell engagers

[0271] One aspect of the present application provides an immune effector cell engager comprising: (a) a target cell binding domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), and (b) an immune effector cell binding domain comprising an antigen-binding fragment that specifically binds to an antigen on an immune effector cell. In some embodiments, the anti-CLL1 sdAb comprises any one of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 57, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60, a CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 64, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO: 74, a CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 78, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO: 81, a CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 85, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a CDR2 comprising the amino acid sequence of SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 92, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO: 151, a CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 155, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO: 158, a CDR2 comprising the amino acid sequence of SEQ ID NO: 160, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 162, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; or (16) a CDR1 comprising the amino acid sequence of SEQ ID NO: 165, a CDR2 comprising the amino acid sequence of SEQ ID NO: 167, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 169, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the anti-CLL1 sdAb comprises a VHH domain comprising the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173. In some embodiments, the antigen-binding fragment in the immune effector cell binding domain is a Fab, scFv, or sdAb. In some embodiments, the target cell binding domain is fused to the N-terminus of the immune effector cell binding domain. In some embodiments, the target cell binding domain is fused to the C-terminus of the immune effector cell binding domain. In some embodiments, the target cell binding domain is fused to the immune effector cell binding domain via a peptide linker. In some embodiments, the immune effector cell binding domain specifically binds to an antigen selected from the group consisting of CD3γ, CD3δ, CD3ε, CD3ζ, CD28, OX40, GITR, CD137, CD27, CD40L, and HVEM.

[0272] In some embodiments, there is provided a T cell engager comprising: (a) a target cell binding domain comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein), and (b) a T cell binding domain comprising an antigen-binding fragment that specifically binds to an antigen on a T cell. In some embodiments, the anti-CLL1 sdAb comprises any one of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 57, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60, a CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 64, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO: 74, a CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 78, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO: 81, a CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 85, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a CDR2 comprising the amino acid sequence of SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 92, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO: 151, a CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 155, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO: 158, a CDR2 comprising the amino acid sequence of SEQ ID NO: 160, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 162, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs; or (16) a CDR1 comprising the amino acid sequence of SEQ ID NO: 165, a CDR2 comprising the amino acid sequence of SEQ ID NO: 167, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 169, or a variant thereof comprising up to about 5 amino acid substitutions in the CDRs. In some embodiments, the anti-CLL1 sdAb comprises a VHH domain comprising the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173, or a variant thereof comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173. In some embodiments, the antigen-binding fragment in the T cell binding domain is a Fab, scFv, or sdAb. In some embodiments, the target cell binding domain is fused to the N-terminus of the T cell binding domain. In some embodiments, the target cell binding domain is fused to the C-terminus of T cell binding domain. In some embodiments, the target cell binding domain is fused to the T cell binding domain via a peptide linker. In some embodiments, the immune effector cell binding domain specifically binds to an antigen selected from the group consisting of CD3γ, CD3δ, CD3ε, CD3ζ, CD28, OX40, GITR, CD137, CD27, CD40L, and HVEM. In some embodiments, the immune effector cell binding domain comprises an antigen-binding fragment that specifically binds to CD3, such as CD3 \varepsilon. Target cell binding domain

[0273] The immune effector cell engagers described herein comprise a target cell binding domain comprising an anti-CLL1 sdAb. In some embodiments, the target cell binding domain consists of an anti- CLL1 sdAb. In some embodiments, the target cell binding domain comprises an anti-CLL1 sdAb and one or more antigen-binding fragments derived from single-domain antibodies or four-chain antibodies that specifically bind to an antigen on a target cell. In some embodiments, the target cell is a tumor cell or a myeloid cell.

[0274] In some embodiments, the target cell binding domain has two or more (such as about any one of 2, 3, 4, 5, 6, or more) antigen binding fragments such as single-domain antibodies. In some embodiments, the multivalent target cell binding domain targets CLL1 only, and comprises two or more antigen binding fragments for CLL1. In some embodiments, the multivalent target cell binding domain targets more than one antigen, and the multivalent target cell binding domain comprises two or more antigen binding fragments for at least one antigen. The antigen binding fragments specific for the same antigen may bind to the same epitope of the antigen or bind to different epitopes of the antigen. The antigen binding fragments specific for the same antigen may comprise the same or different single-domain antibodies.

[0275] In some embodiments, the target cell binding domain comprises a plurality of anti-CLL1 sdAbs. In some embodiments, the plurality of the anti-CLL1 sdAb is fused to each other via peptide bonds or peptide linkers. In some embodiments, each peptide linker is no more than about 50 (such as no more than about any one of 35, 25, 20, 15, 10, or 5) amino acids long.

[0276] In some embodiments, the target cell binding domain can specifically bind to two or more (such as about any one of 2, 3, 4, 5, 6, or more) different antigens. In some embodiments, the multispecific target cell binding domain has one antigen binding fragments for each antigen. In some embodiments, the multispecific target cell binding domain has more than two antigen binding fragments for at least one antigen. Each antigen binding fragment may comprise a single-domain antibody.

[0277] Depending on the desired antigens to be targeted, the target cell binding domain can be engineered to include the appropriate single-domain antibodies that are specific to the desired antigens. In some embodiments, the target cell binding domain comprises an anti-CLL1 sdAb and an anti-CD33 sdAb. The antigen binding fragments (such as sdAbs) can be arranged in any suitable order. For example, a first sdAb is fused to the N-terminus or the C-terminus of a second sdAb. A suitable peptide linker may be placed between different sdAbs to avoid steric hindrance between the sdAbs. Immune effector cell binding domain

[0278] The immune effector cell engagers described herein comprise an immune effector cell binding domain. The immune effector cell binding domain comprises an antigen-binding fragment that specifically binds to an antigen on an immune effector cell. Immune effector cells include, but are not limited to, T cells and NK cells.

[0279] In some embodiments, the immune effector cell binding domain specifically binds to CD3, such as human CD3. "CD3" is known in the art as a multi-protein complex of six chains (see, Abbas and Lichtman, 2003; Janeway et al., p172 and 178, 1999). In mammals, the complex comprises a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, and a homodimer of CD3 zeta chains. CD3 as used herein may be from various animal species, including human, primate, mouse, rat, or other mammals. In some embodiments, the immune effector cell binding domain comprises an antigen-binding fragment that specifically binds to an individual CD3 chain, such as CD3 gamma chain, CD3 delta chain, or CD3 epsilon chain. In some embodiments, the antigen-binding fragment specifically binds to a complex formed from two or more individual CD3 chains (e.g., a complex of more than one CD3 epsilon chains, a complex of a CD3 gamma and CD3 epsilon chain, a complex of a CD3 delta and CD3 epsilon chain). In some embodiments, the antigen-binding fragment specifically binds to a CD3 epsilon chain.

[0280] The antigen-binding fragment targeting CD3 can be of any suitable antigen-binding fragments, including but not limited to Fab, scFv, and sdAb (e.g., VHH). In some embodiments, the antigen-binding fragment is murine, camelid, chimeric, human or humanized. The antigen-binding fragment can be designed based on any known CD3 antibodies in the art, including, but not limited to, SP34 mouse monoclonal antibody, (see, for example, Pressano, S. The EMBO J. 4:337-344, 1985; Alarcon, B. EMBO J. 10:903- 912, 1991; Salmeron A. et al., J. Immunol. 147:3047-52, 1991; Yoshino N. et al., Exp. Anim 49:97-110, 2000; Conrad M L. et al., Cytometry 71A:925-33, 2007; and Yang et al., J. Immunol. 137:1097-1100: 1986), Cris-7 monoclonal antibody (Reinherz, E. L. et al. (eds.), Leukocyte typing II, Springer Verlag, New York, (1986)), BC3 monoclonal antibody (Anasetti et al. (1990) J. Exp. Med. 172:1691), OKT3 (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med. 313:337) and derivatives thereof such as OKT3 ala-ala (Herold et al. (2003) J. Clin. Invest. 11:409), visilizumab (Carpenter et al. (2002) Blood 99:2712), 145-2C11 monoclonal antibody (Hirsch et al. (1988) J. Immunol. 140: 3766), UCHT-1 (Beverley, P C and Callard, R. E. (1981) Eur. J. Immunol. 11: 329-334), anti-CD3 sdAbs (such as 60E11 and 117G03) described in WO2016180982, and CD3 binding molecules described in WO2004 / 106380; WO2004 / 106381; WO2010 / 037838; WO2008 / 119567; WO2007 / 042261; WO2010 / 0150918. In some embodiments, the anti-CD3 antigen-binding fragment is derived from OKT3, L2K, UCHT1, 60E11 or 117G03. In some embodiments, the anti-CD3 antigen-binding fragment is an scFv derived from OKT3, L2K or UCHT. In some embodiments, the anti-CD3 antigen-binding fragment is a VHH derived from 60E11 or 117G03. In some embodiments, the anti-CD3 antigen-binding fragment is derived from an antibody that binds to the same epitope as OKT3, L2K, UCHT1, 60E11 or 117G03. In some embodiments, the anti-CD3 antigen-binding fragment is derived from an antibody that specifically binds to CD3 competitively with OKT3, L2K, UCHT1, 60E11 or 117G03. Signal peptide

[0281] The immune effector cell engagers of the present application may comprise a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. In general, signal peptides are peptide sequences that target a polypeptide to the desired site in a cell. In some embodiments, the signal peptide targets the immune effector cell engager to the secretory pathway of the cell and will allow secretion of the immune effector cell engager into the cell culture media. Signal peptides including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences. In some embodiments, the signal peptide is derived from a human albumin signal peptide. In some embodiments, the signal peptide is derived from a human azurocidin secretion signal. Peptide linkers

[0282] The target cell binding domain and the immune effector cell binding domain may be fused to each other via a peptide linker. In some embodiments, the target cell binding domain and the immune effector cell binding domain are directly fused to each other without any peptide linker.

[0283] In some embodiments, the various antigen-binding fragments (such as sdAbs) in the multispecific or multivalent target cell binding domain are fused to each other via peptide linker(s). In some embodiments, the antigen-binding fragments (such as sdAbs) are directly fused to each other without any peptide linkers. The peptide linkers connecting different antigen-binding fragments (such as sdAbs) may be the same or different.

[0284] Each peptide linker in an immune effector cell engager may have the same or different length and / or sequence depending on the structural and / or functional features of the antigen-binding fragments (such as sdAbs) and / or the various domains. Each peptide linker may be selected and optimized independently. The length, the degree of flexibility and / or other properties of the peptide linker(s) used in the immune effector cell engagers may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes. For example, longer peptide linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another. For example, in a multivalent or multispecific target cell binding domain that comprises sdAbs directed against a multimeric antigen, the length and flexibility of the peptide linkers are preferably such that it allows each antigen-binding fragment (such as sdAb) to bind to the antigenic determinant on each of the subunits of the multimer.

[0285] In some embodiment, a peptide linker comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other. For example, a (GGGGS)3 linker (SEQ ID NO: 141) can be a suitable peptide linker between the target cell binding domain and the immune effector cell binding domain. In some embodiments, the peptide linker is no more than about 50 (such as no more than about any one of 35, 25, 20, 15, 10, or 5) amino acids long.

[0286] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids long. In some embodiments, the peptide linker is no more than about any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids long. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acids to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0287] The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGGS)n, and (GGGGS)n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, the peptide linker comprises the amino acid sequence GGGGS (SEQ ID NO: 143), (GGGGS)2 (SEQ ID NO: 144), (GGGGS)3 (SEQ ID NO: 141), (GGGGS)4 (SEQ ID NO: 182), (GGGGS)5 (SEQ ID NO: 183), (GGGS)2 (SEQ ID NO: 145), (GGGS)4 (SEQ ID NO: 146), or GSTSGSGKPGSGEGSTKG (SEQ ID NO: 147). D. Immunoconjugates

[0288] In one aspect, the present application provides immunoconjugates comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein) and an effector molecule. Exemplary effector molecules include, but are not limited to, a drug, a toxin, a radioisotope, a protein, a peptide, a nucleic acid, and a label.

[0289] In some embodiments, there is provided an immunoconjugate comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein) conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

[0290] In some embodiments, an immunoconjugate is an antibody-drug conjugate (ADC) in which an anti-CLL1 sdAb is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

[0291] In some embodiments, an immunoconjugate comprises an anti-CLL1 sdAb as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[0292] In some embodiments, an immunoconjugate comprises an anti-CLL1 sdAb as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, "MRI"), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0293] Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94 / 11026. The linker may be a "cleavable linker" facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0294] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4- vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

[0295] In some embodiments, any of the anti-CLL1 sdAbs provided herein is useful for detecting the presence of CLL1 in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample is blood, serum or other liquid samples of biological origin. In some embodiments, a biological sample comprises a cell or tissue.

[0296] In some embodiments, the present application provides an immunoconjugate comprising an anti-CLL1 sdAb (such as any one of the anti-CLL1 sdAbs described herein) and a label. In some embodiments, the label is conjugated to the anti-CLL1 sdAb. In some embodiments, there is provided a method of detecting CLL1 in a cell, comprising contacting the cell with the immunoconjugate. In some embodiments, a method of detecting the presence of CLL1 in a biological sample is provided. In some embodiments, the method comprises detecting the presence of CLL1 protein in a biological sample. In some embodiments, the CLL1 is human CLL1. In some embodiments, the method comprises contacting the biological sample with the immunoconjugate under conditions permissive for binding of the anti- CLL1 sdAb to CLL1, and detecting signal from the label. Such method may be an in vitro or in vivo method. In some embodiments, there is provided a method of diagnosing a disease associated with CLL1 expression (e.g., acute myeloid leukemia) in an individual, comprising administering to the individual the immunoconjugate, and detecting the label in the individual. In some embodiments, the immunoconjugate is used to select subjects eligible for therapy with any of the anti-CLL1 therapeutic agents described herein (e.g., anti-CLL1 sdAb, chimeric receptor, immune effector cell engager, and engineered immune cell), wherein CLL1 is a biomarker for selection of patients.

[0297] In some embodiments, labeled anti-CLL1 sdAbs are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3- dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6- phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like. E. Features of antibody moieties

[0298] In some embodiments, any antibody moiety in the anti-CLL1 constructs described herein may incorporate any of the features, singly or in combination, as described in sections 1-7 below. 1. Antibody Affinity

[0299] In some embodiments, an antibody moiety provided herein has a dissociation constant (Kd) of <semantics>≤1μM,≤100nM,≤10nM,≤1nM,≤0.1nM,≤0.01nM,or≤0.001nM(e.g.10−8Morless,e.g.from<annotation encoding="application / x-tex">\leq 1 \,\mu\text{M}, \leq 100 \,\text{nM}, \leq 10 \,\text{nM}, \leq 1 \,\text{nM}, \leq 0.1 \,\text{nM}, \leq 0.01 \,\text{nM}, \text{or} \leq 0.001 \,\text{nM} \,(e.g. \, 10^{-8} \,\text{M} \,\text{or} \, \text{less}, \, e.g. \, \text{from}< / annotation>< / semantics> <semantics>10−8<annotation encoding="application / x-tex">10^{-8}< / annotation>< / semantics> M to <semantics>10−13<annotation encoding="application / x-tex">10^{-13}< / annotation>< / semantics> M, e.g., from <semantics>10−9<annotation encoding="application / x-tex">10^{-9}< / annotation>< / semantics> M to <semantics>10−13<annotation encoding="application / x-tex">10^{-13}< / annotation>< / semantics> M).

[0300] In some embodiments, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version or VHH fragment of an antibody of interest and its antigen as described by the following assay. For example, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)).

[0301] In some embodiments, Kd is measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at <semantics>∼10<annotation encoding="application / x-tex">\sim 10< / annotation>< / semantics> response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 µg / ml (~0.2 µM) before injection at a flow rate of 5 µl / minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab or VHH of the antibody of interest (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20TM) surfactant (PBST) at 25°C at a flow rate of approximately 25 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>l / min. Association rates (<semantics>kon<annotation encoding="application / x-tex">k_{on}< / annotation>< / semantics>) and dissociation rates (<semantics>koff<annotation encoding="application / x-tex">k_{off}< / annotation>< / semantics>) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio <semantics>koff / kon<annotation encoding="application / x-tex">k_{off} / k_{on}< / annotation>< / semantics>. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds <semantics>106M−1s−1<annotation encoding="application / x-tex">10^6 \, \text{M}^{-1} \, \text{s}^{-1}< / annotation>< / semantics> by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20 nM anti-antigen antibody moiety in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCOTM spectrophotometer (ThermoSpectronic) with a stirred cuvette. 2. Antibody Fragments

[0302] In some embodiments, an antibody moiety provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, VHH, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half- life, see U.S. Patent No. 5,869,046.

[0303] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0304] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein. 3. Chimeric and Humanized Antibodies

[0305] In some embodiments, an antibody moiety provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a camelid species, such as llama) and a human constant region. In a further example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen- binding fragments thereof.

[0306] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0307] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36...

Claims

1. An anti-CLL1 construct comprising a single domain antibody (sdAb) moiety that specifically binds to CLL1 (anti-CLL1 sdAb moiety), wherein the anti-CLL1 sdAb moiety comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 29; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 36; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 50; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 57; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60, a CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 64; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 71; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO: 74, a CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 78; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO: 81, a CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 85; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a CDR2 comprising the amino acid sequence of SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 92; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO: 151, a CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 155; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO: 158, a CDR2 comprising the amino acid sequence of SEQ ID NO: 160, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 162; or (16) a CDR1 comprising the amino acid sequence of SEQ ID NO: 165, a CDR2 comprising the amino acid sequence of SEQ ID NO: 167, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 169.

2. An anti-CLL1 construct comprising a single domain antibody moiety that specifically binds to CLL1 (anti-CLL1 sdAb moiety), wherein the anti-CLL1 sdAb moiety comprises a CDR1, a CDR2, and a CDR3 of an anti-CLL1 sdAb comprising the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173.

3. The anti-CLL1 construct of claim 1 or 2, wherein the anti-CLL1 sdAb moiety comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173.

4. The anti-CLL1 construct of claim 3, wherein the anti-CLL1 sdAb moiety comprises the amino acid sequence of any one of SEQ ID NOs: 94-106 and 171-173.

5. The anti-CLL1 construct of any one of claims 1-4, wherein the anti-CLL1 construct comprises an extracellular domain comprising the anti-CLL1 sdAb moiety, a transmembrane domain, and an intracellular signaling domain comprising a primary intracellular signaling sequence of an immune effector cell.

6. The anti-CLL1 construct of claim 5, wherein the primary intracellular signaling sequence is derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d.

7. The anti-CLL1 construct of claim 5 or 6, wherein the intracellular signaling domain further comprises an intracellular co-stimulatory sequence.

8. The anti-CLL1 construct of claim 5 or 6, wherein the intracellular signaling domain does not comprise an intracellular co-stimulatory sequence.

9. The anti-CLL1 construct of any one of claims 1-4, wherein the anti-CLL1 construct comprises an extracellular domain comprising the anti-CLL1 sdAb moiety, a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence.

10. The anti-CLL1 construct of claim 7 or 9, wherein the intracellular co-stimulatory sequence is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof.

11. An anti-CLL1 construct comprising an extracellular domain comprising a single domain antibody moiety that specifically binds to CLL1 (anti-CLL1 sdAb moiety), a transmembrane domain, and an intracellular signaling domain, wherein the anti-CLL1 sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43.

12. The anti-CLL1 construct of any one of claims 5-11, wherein the extracellular domain of the anti- CLL1 construct further comprises a second binding moiety that specifically binds to a second antigen or epitope.

13. The anti-CLL1 construct of claim 12, wherein the second binding moiety is an sdAb, an scFv, or an extracellular domain of a receptor.

14. The anti-CLL1 construct of claim 13, wherein the second binding moiety is an sdAb or scFv that specifically binds to CD33 or CD123.

15. The anti-CLL1 construct of claim 13, wherein the second binding moiety is an extracellular domain of NKG2D.

16. An anti-CLL1 construct comprising an extracellular domain comprising a single domain antibody moiety that specifically binds to CLL1 (anti-CLL1 sdAb moiety) and an sdAb that specifically binds to CD33 (anti-CD33 sdAb moiety), a transmembrane domain, and an intracellular signaling domain, wherein the anti-CLL1 sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43, and wherein the anti-CD33 sdAb moiety comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 198, a CDR2 comprising the amino acid sequence of SEQ ID NO: 200, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 202.

17. An anti-CLL1 construct comprising the amino acid sequence of any one of SEQ ID NOs: 120- 132, 177-179, 181, 184-195, 229-230, and 234-236.

18. A nucleic acid encoding the anti-CLL1 construct of any one of claims 1-17.

19. An engineered immune cell comprising the anti-CLL1 construct of any one of claims 1-17, or the nucleic acid of claim 18.

20. The engineered immune cell of claim 19, further comprising a second chimeric receptor.

21. The engineered immune cell of claim 20, wherein the second chimeric receptor comprises an extracellular domain comprising a second binding moiety that specifically binds to a second antigen or epitope, a transmembrane domain, and an intracellular signaling domain comprising a primary intracellular signaling sequence of an immune effector cell.

22. The engineered immune cell of claim 21, wherein the intracellular signaling domain further comprises an intracellular co-stimulatory sequence.

23. The engineered immune cell of claim 20, wherein the second chimeric receptor comprises an extracellular domain comprising a second binding moiety that specifically binds to a second antigen or epitope, a transmembrane domain, and an intracellular signaling domain comprising an intracellular co-stimulatory sequence.

24. The engineered immune cell of any one of claims 21-23, wherein the second binding moiety is an sdAb, an scFv, or an extracellular domain of a receptor.

25. The engineered immune cell of claim 24, wherein the second binding moiety is an sdAb or scFv that specifically binds to CD33 or CD123.

26. The engineered immune cell of claim 24, wherein the second binding moiety is an extracellular domain of NKG2D.

27. An engineered immune cell comprising: (a) a first chimeric receptor comprising an extracellular domain comprising an anti-CLL1 sdAb, a transmembrane, and an intracellular signaling domain, wherein the anti-CLL1 sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8; or (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and (b) a second chimeric receptor comprising an extracellular domain comprising an anti-CD33 sdAb, a transmembrane domain, and an intracellular signaling domain, wherein the anti-CD33 sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 205, a CDR2 comprising the amino acid sequence of SEQ ID NO: 207, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 209; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 212, a CDR2 comprising the amino acid sequence of SEQ ID NO: 214, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 216; or (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 219, a CDR2 comprising the amino acid sequence of SEQ ID NO: 221, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223.

28. The engineered immune cell of any one of claims 19-27, wherein the immune cell is a T cell.

29. The engineered immune cell of claim 28, wherein the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a γδT cell.

30. The engineered immune cell of any one of claims 19-29, wherein the engineered immune cell expresses a safety-switch antigen or epitope.

31. The engineered immune cell of claim 30, wherein the safety-switch antigen or epitope is derived from CD52, EGFR or CD20.

32. The anti-CLL1 construct of any one of claims 1-4, wherein the anti-CLL1 construct is a monospecific molecule.

33. The anti-CLL1 construct of any one of claims 1-4, wherein the anti-CLL1 construct is a multispecific molecule.

34. The anti-CLL1 construct of claim 33, wherein the anti-CLL1 construct comprises the sdAb moiety linked to a second binding moiety that specifically binds to a second antigen or epitope.

35. The anti-CLL1 construct of claim 34, wherein the second binding moiety is an sdAb or an scFv.

36. The anti-CLL1 construct of claim 34 or 35, wherein the sdAb moiety is linked to the second binding moiety via a peptide linker.

37. The anti-CLL1 construct of any one of claims 34-36, wherein the second binding moiety specifically binds to an antigen on the surface of a T cell.

38. The anti-CLL1 construct of claim 37, wherein the second binding moiety specifically binds to an antigen selected from the group consisting of CD3γ, CD3δ, CD3ε, CD3ζ, CD28, OX40, GITR, 4-1BB, CD27, CD40L, and HVEM.

39. The anti-CLL1 construct of any one of claims 1-4, wherein the anti-CLL1 construct is an immunoconjugate comprising the sdAb moiety and an effector molecule.

40. The anti-CLL1 construct of claim 39, wherein the effector molecule is a therapeutic agent selected from the group consisting of a drug, a toxin, a radioisotope, a protein, a peptide, and a nucleic acid.

41. The anti-CLL1 construct of claim 39, wherein the effector molecule is a drug or a toxin.

42. The anti-CLL1 construct of claim 39, wherein the effector molecule is a label.

43. The anti-CLL1 construct of any one of claims 1-4, wherein the anti-CLL1 construct is monoclonal antibody, single-domain antibody or full-length heavy-chain only antibody.

44. A nucleic acid encoding the anti-CLL1 construct of claim 43.

45. A vector comprising the nucleic acids of claim 18 or 44.

46. A pharmaceutical composition comprising (1) the anti-CLL1 construct of any one of claims 1-17 and 32-43 or the engineered immune cell of any one of claims 19-31; and (2) a pharmaceutically acceptable carrier.

47. Use of the anti-CLL1 construct of any one of claims 1-17 and 32-43, the engineered immune cell of any one of claims 19-31, or the pharmaceutical composition of claim 46 in the manufacture of a medicament for treating an individual having a cancer.

48. The use of claim 47, wherein the cancer is acute myeloid leukemia (AML), chronic myeloid leukemia (CML) or myelodysplastic syndrome (MDS).