CLL-1 / CD33 dual-targeting chimeric antigen receptors

Abstract

The present disclosure provides CLL-1 / CD33 dual-targeting chimeric antigen receptors (CARs). The CARs may comprise a VHH targeting CLL-1 and a VHH targeting CD33. The chimeric antigen receptors can be used to treat cancer, such as acute myeloid leukemia (AML).

Description

[0001] CLL-1 / CD33 DUAL-TARGETING CHIMERIC ANTIGEN RECEPTORS

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] The present application claims priority to U.S. Provisional Patent Application No.

[0004] 63 / 791,024 filed on April 18, 2025, which is hereby incorporated by reference in its entirety.

[0005] INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0006] A sequence listing, filed as the XML file “11299_013170-WO0_SL.xml” which was created on April 13, 2026 and is 657,210 bytes in size, is incorporated herein by reference in its entirety.

[0007] FIELD OF THE INVENTION

[0008] The present disclosure relates to the field of immunotherapy, and more particularly to CLL-1 / CD33 dual-targeting chimeric antigen receptors (CARs).

[0009] BACKGROUND

[0010] Acute myeloid leukemia (AML) is the most common acute leukemia in adults, characterized by the clonal proliferation of malignant myeloid blast cells in bone marrow along with impaired normal hematopoiesis (De Kouchkovsky et al., Acute myeloid leukemia: a comprehensive review and 2016 update, Blood Cancer J. 6, e441 (2016); Saif et al., Acute Myeloid Leukemia: Is That All There Is? Cureus 10, e3198 (2018)). To date, a wide variety of therapeutic strategies have been developed for AML including allogeneic hematopoietic stem cell transplantation (alloHSCT), chemotherapy, target therapy, and immunotherapy-based treatments (Marofi et al., Novel CAR T therapy is a ray of hope in the treatment of seriously ill AML patients. Stem Cell Res. Ther. 12, 465 (2021)). However, the prognosis remains poor and treatment options for relapsed AML are very limited (Koenig et al., The Changing Landscape of Treatment in Acute Myeloid Leukemia, Am. Soc. Clin. Oncol. Educ. Book 40, 1-12 (2020)).

[0011] The advent of chimeric antigen receptor (CAR) T-cell therapy has led to dramatic remission rates in multiple relapsed / refractory (r / r) hematologic malignancies (June et al., Chimeric Antigen Receptor Therapy, N. Engl. J. Med. 379, 64-73 (2018)). While CAR T-cell therapy has been particularly successful for B-cell malignancies, effectively treating AML withCARs has posed a larger challenge. AML not only creates an immunosuppressive tumor microenvironment that dampens CAR-T cell responses, but also lacks many unique tumor-associated antigens (TAAs), making leukemic-specific targeting difficult. Since many AML targets under investigation are also expressed on hematopoietic stem cells (HSCs), CAR-T cell therapy can lead to severe and potentially lethal myeloablation (Marvin-Peek et al., Challenges and Advances in Chimeric Antigen Receptor Therapy for Acute Myeloid Leukemia. Cancers (Basel) 14 (2022)). For example, CD33 is expressed on AML blast cells and leukemia stem cells (LSCs) in most patients, but it is also present on HSCs and widely distributed in liver tissue with a high expression level on hepatocytes (Maniecki et al., Is hepatotoxicity in patients treated with gemtuzumabozogamicin due to specific targeting of hepatocytes? Leuk. Res. 35, e84-86 (2011)), which raises many concerns on CD33-targeting associated adverse effects. Therefore, CAR-T cell therapy works well for B-cell malignancies, but successful translation to AML is yet to be accomplished. At present, no CAR-T cell therapy for the treatment of AML has been approved by FDA.

[0012] There is an urgent need to develop methods to effectively treat AML.

[0013] SUMMARY

[0014] The present disclosure provides a nucleic acid comprising a first polynucleotide and a second polynucleotide.

[0015] The present disclosure also provides a system comprising a first polynucleotide and a second polynucleotide.

[0016] The first polynucleotide and the second polynucleotide may be on a single nucleic acid molecule, or on separate nucleic acid molecules.

[0017] The first polynucleotide encodes a first polypeptide, where the first polypeptide comprises an anti-CLL- 1 antigen-binding region.

[0018] The present disclosure provides a recombinant polypeptide. The recombinant polypeptide may comprise an anti-CLL- 1 antigen-binding region. The recombinant polypeptide may comprise an anti-CLL- 1 VHH.

[0019] The anti-CLL- 1 antigen-binding region may comprise a (first) VHH (an anti-CLL- 1 VHH).

[0020] The second polynucleotide encodes a second polypeptide, where the second polypeptidecomprises an anti-CD33 antigen-binding region.

[0021] The present disclosure provides a recombinant polypeptide. The recombinant polypeptide may comprise an anti-CD33 antigen-binding region. The recombinant polypeptide may comprise an anti-CD33 VHH.

[0022] The anti-CD33 antigen-binding region may comprise a (second) VHH (an anti-CD33 VHH).

[0023] The (first) VHH (the anti-CLL- 1 VHH) may comprise three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, respectively; (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively; (4) SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, respectively; (5) SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, respectively; (6) SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, respectively; (7) SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, respectively; (8) SEQ ID NO: 102. SEQ ID NO: 104, SEQ ID NO: 106, respectively; (9) SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, respectively; (10) SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, respectively; (11) SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, respectively; or (12) SEQ ID NO: 198, SEQ ID NO:200, SEQ ID NO:202, respectively.

[0024] The (first) VHH (the anti-CLL- 1 VHH) may comprise three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74. respectively; (2) SEQ ID NO: 118, SEQ ID NO: 120. SEQ ID NO: 122, respectively; or (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively.

[0025] The (first) VHH (the anti-CLL- 1 VHH) may comprise an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO:115, SEQ ID NO:147, SEQ ID NO:19, SEQ ID NO:35, SEQ ID NO:51, SEQ ID NO:83, SEQ ID NO:99, SEQ ID NO: 131, SEQ ID NO: 163, SEQ ID NO: 179, or SEQ ID NO: 195.

[0026] The (first) VHH (the anti-CLL- 1 VHH) may comprise an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, or SEQ ID NO: 147.

[0027] The (second) VHH (the anti-CD33 VHH) may comprise three complementaritydetermining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively; (4) SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, respectively; (5) SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, respectively; (6) SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298. respectively; (7) SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, respectively; (8) SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, respectively; (9) SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, respectively; (10) SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, respectively; or (11) SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, respectively.

[0028] The (second) VHH (the anti-CD33 VHH) may comprise three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; or (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively.

[0029] The (second) VHH (the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, SEQ ID NO:419, SEQ ID NO:259, SEQ ID NO:275, SEQ ID NO: 291, SEQ ID NO:323, SEQ ID NO:355, SEQ ID NO:371, SEQ ID NO:387. or SEQ ID NO:403.

[0030] The (second) VHH (the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, or SEQ ID NO:419.

[0031] In certain embodiments, the first polypeptide is a first chimeric antigen receptor (CAR) (component), and the second polypeptide is a second CAR (component).

[0032] In certain embodiments, the first polypeptide further comprises a first hinge region, a first transmembrane domain, or a combination thereof. In certain embodiments, the second polypeptide further comprises a second hinge region, a second transmembrane domain, or a combination thereof. The first hinge region may be different from, or the same as, the second hinge region. The first transmembrane domain may be different from, or the same as, the second transmembrane domain.

[0033] In certain embodiments, the first polypeptide further comprises a first cytoplasmicsignaling domain, and the second polypeptide further comprises a second co-stimulatory region. In certain embodiments, the first polypeptide further comprises a first co-stimulatory region, and the second polypeptide further comprises a second cytoplasmic signaling domain.

[0034] The first polynucleotide may be located at the 5’ end of the second polynucleotide.

[0035] The second polynucleotide may be located at the 5’ end of the first polynucleotide.

[0036] In certain embodiments, the first polypeptide comprises, from N-terminus to C-terminus: the first VHH, a first hinge region, a first transmembrane domain, and a first cytoplasmic signaling domain.

[0037] In certain embodiments, the second polypeptide comprises, from N-terminus to C-terminus: the second VHH, a second hinge region, a second transmembrane domain, and a second co-stimulatory region.

[0038] The first polypeptide and the second polynucleotide may be linked by a linker. The linker may be a cleavable linker. The cleavable linker may be a self-cleaving peptide, such as a 2A peptide (e.g.. P2A, E2A, T2A, or F2A). The linker may be a non-cleavable linker.

[0039] In certain embodiments, the first polypeptide further comprises a first signal peptide. In certain embodiments, the second polypeptide further comprises a second signal peptide. The first signal peptide may be the same as, or different from, the second signal peptide.

[0040] In certain embodiments, the first polynucleotide comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:572, SEQ ID NO:574, SEQ ID NO:532, SEQ ID NO:534, SEQ ID NO: 482, SEQ ID NO:484, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:502, SEQ ID NO:504, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:522, SEQ ID NO:524, SEQ ID NO:542. SEQ ID NO:544, SEQ ID NO:552, SEQ ID NO:554, SEQ ID NO:562, or SEQ ID NO:564.

[0041] In certain embodiments, the first polynucleotide comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:572, SEQ ID NO:574, SEQ ID NO:532, or SEQ ID NO:534.

[0042] In certain embodiments, the second polynucleotide comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:576, SEQ ID NO:578, SEQ ID NO:536, SEQ ID NO:538, SEQ ID NO:486, SEQ ID NO:488, SEQ ID NO:496, SEQ ID NO:498, SEQ ID NO:506. SEQ ID NO:508, SEQ ID NO:516, SEQ ID NO:518, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:546, SEQ ID NO:548, SEQ ID NO:556, SEQ ID NO:558,SEQ ID NO:566, or SEQ ID NO:568.

[0043] In certain embodiments, the second polynucleotide comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:576, SEQ ID NO:578, SEQ ID NO:536, or SEQ ID NO:538.

[0044] In certain embodiments, the first polypeptide comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:571, SEQ ID NO:573, SEQ ID NO:531, SEQ ID NO:533, SEQ ID NO:481, SEQ ID NO:483, SEQ ID NO:491, SEQ ID NO:493, SEQ ID NO:501, SEQ ID NO:503, SEQ ID NO:511, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:523, SEQ ID NO:541, SEQ ID NO:543, SEQ ID NO:551, SEQ ID NO:553, SEQ ID NO:561, or SEQ ID NO:563.

[0045] In certain embodiments, the first polypeptide comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:571, SEQ ID NO:573, SEQ ID NO:531, or SEQ ID NO:533.

[0046] In certain embodiments, the second polypeptide comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:575, SEQ ID NO:577, SEQ ID NO:535, SEQ ID NO:537, SEQ ID NO:485, SEQ ID NO:487, SEQ ID NO:495, SEQ ID NO:497, SEQ ID NO:505, SEQ ID NO:507, SEQ ID NO:515, SEQ ID NO:517, SEQ ID NO:525, SEQ ID NO:527, SEQ ID NO:545, SEQ ID NO:547, SEQ ID NO:555, SEQ ID NO:557, SEQ ID NO:565, or SEQ ID NO:567.

[0047] In certain embodiments, the second polypeptide comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:575, SEQ ID NO:577, SEQ ID NO:535, or SEQ ID NO:537.

[0048] In certain embodiments, the nucleic acid comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:570, SEQ ID NO:530, SEQ ID NO:480, SEQ ID NO:490, SEQ ID NO:500, SEQ ID NO:510, SEQ ID NO:520, SEQ ID NO:540, SEQ ID NO:550, or SEQ ID NO:560.

[0049] In certain embodiments, the nucleic acid comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:570, or SEQ ID NO:530.

[0050] In certain embodiments, the nucleic acid encodes a polypeptide comprising an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ IDNO:569, SEQ ID NO:529, SEQ ID NO:479, SEQ ID NO:489, SEQ ID NO:499, SEQ ID NO:509, SEQ ID NO:519, SEQ ID NO:539, SEQ ID NO:549, or SEQ ID NO:559.

[0051] In certain embodiments, the nucleic acid encodes a polypeptide comprising an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:569, or SEQ ID NO:529.

[0052] In certain embodiments, the nucleic acid encodes a multi-specific (e.g„ bispecific) antibody, where the first polypeptide is a first antibody (fragment), and wherein the second polypeptide is a second antibody (fragment).

[0053] In certain embodiments, the recombinant polypeptide comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO: 225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO: 235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO: 245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251. SEQ ID NO:253, SEQ ID NO:255, or SEQ ID NO:257.

[0054] In certain embodiments, the recombinant polypeptide comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:435, SEQ ID NO:437, SEQ ID NO:439, SEQ ID NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ ID NO:449, SEQ ID NO:451, SEQ ID NO:453, SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459, SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465, SEQ ID NO:467, SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID NO:475, or SEQ ID NO:477.

[0055] In certain embodiments, the recombinant polypeptide is a chimeric antigen receptor (CAR) or an antibody.

[0056] The first polypeptide, the second polypeptide, or the recombinant polypeptide, may be a chimeric antigen receptor (CAR). The CAR may further comprise one or more of the following:

[0057] (a) a signal peptide,

[0058] (b) a hinge region,

[0059] (c) a transmembrane domain,

[0060] (d) a co- stimulatory region, and(e) a cytoplasmic signaling domain.

[0061] The co-stimulatory region may comprise a co- stimulatory region of 4- IBB (CD 137), CD28, 0X40, CD2, CD7, CD27, CD30, CD40, CD70, CD134, PD1, DaplO, CDS, ICAM-1, LFA-1 (CDlla / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

[0062] The cytoplasmic signaling domain may comprise a cytoplasmic signaling domain of CD3 . The hinge region may comprise a hinge region of IgG4, CD8, CD28, CD137, or combinations thereof.

[0063] The transmembrane domain may comprise a transmembrane domain of CD8, CD28, CD3s, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154, or combinations thereof.

[0064] The recombinant polypeptide may be an antibody. The recombinant polypeptide may further comprise a human Fc region.

[0065] Also encompassed by the present disclosure is a nucleic acid encoding the recombinant polypeptide.

[0066] In certain embodiments, the nucleic acid comprises a nucleotide sequence about 80% to about 100% identical, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%. at least or about 87%, at least or about 88%, at least or about 89%. at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, to the nucleotide sequence set forth in SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO: 226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO: 236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO: 246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, or SEQ ID NO:258.

[0067] In certain embodiments, the nucleic acid comprises a nucleotide sequence about 80% to about 100%, at least or about 70%. at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%. at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least orabout 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%. or about 100%, identical to the nucleotide sequence set forth in SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ ID NO:450, SEQ ID NO:452, SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458, SEQ ID NO:460, SEQ ID NO:462. SEQ ID NO:464. SEQ ID NO:466, SEQ ID NO:468, SEQ ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ ID NO:476, or SEQ ID NO:478.

[0068] The present disclosure also provides a vector comprising the present nucleic acid.

[0069] The present disclosure provides an immune cell. The immune cell may comprise the present nucleic acid. The immune cell may comprise the present vector. The immune cell may comprise, or express, the first polypeptide and the second polypeptide. The immune cell may comprise, or express, the present recombinant polypeptide.

[0070] The immune cell may be a T cell or a natural killer (NK) cell.

[0071] Also encompassed by the present disclosure is a pharmaceutical composition. The pharmaceutical composition may comprise the present immune cell. The pharmaceutical composition may comprise the present nucleic acid. The pharmaceutical composition may comprise the present vector. The pharmaceutical composition may comprise the present recombinant polypeptide.

[0072] The present disclosure provides a method of treating cancer in a subject. The method may comprise administering the immune cell to the subject. The method may comprise administering the pharmaceutical composition to the subject.

[0073] The cancer may be a hematologic cancer. The cancer may be lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, leukemia, and / or multiple myeloma. The cancer may be acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), or combinations thereof.

[0074] The present disclosure provides a method of treating an immunological disease in a subject. The present disclosure provides a method of treating an autoimmune disorder in a subject. The method may comprise administering the immune cell to the subject. The method may comprise administering the pharmaceutical composition to the subject.The autoimmune disorder may be systemic lupus erythematosus (SLE), systemic sclerosis, inflammatory myopathy, systemic scleroderma, multiple sclerosis, myasthenia gravis, a myositis autoantibody-driven disease, or neuromyelitis optica. The inflammatory myopathy may be polymyositis, dermatomyositis, or inclusion-body myositis. The subject with SLE may have lupus nephritis.

[0075] The immune cell may be allogeneic or autologous.

[0076] BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figures 1A-1E. Design, structure and functional verification of CLL-l / CD33-specific dual-CAR constructs. (A) Split duaLCAR strategy is used for designing T cells to attack tumor cells and spare normal tissues (Hanada et al., Double or nothing on cancer immunotherapy, Nat. Biotechnol. 31, 33-34 (2013)). (B-C) Domain structure of an embodiment of the CLL-1 / CD33-specific dual-CAR constructs. H, hinge; TM, transmembrane, SP, signal peptide. (D-E) Differential potency of tested dual-CARs against single antigen vs dual antigens was evaluated by co-culturing T cells and wild type (WT), CLL-1 / CD33 single knockout (sKO), and double knockout (dKO) AML cells (U-937). IFNy release in co-culture supernatants (D) and killing ability (E) were quantitated. Mono-specific CARs (CLL-sCAR and CD33-sCAR) were used as controls to confirm comparative levels of antigen expression on sKO and WT AML cells. RLU (relative light unit) was positively correlated with the number of live cells in luciferase killing assays.

[0078] Figures 2A-2D. CLL-l / CD33-specific dual-CAR T cells (D3 and D4) produce large amounts of IFNy upon both dual targeting and single targeting. (A) Schematic diagrams show the structures of CLL-1- specific first-generation CAR (DI), CD33-specific 4-1BB endo-domain only CAR (D2), CLL-l / CD33-specific dual-CARs with the linking of DI and D2 by P2A peptide (D1-P2A-D2 for D3, and D2-P2A-D1 for D4). (B) Representative flow profile shows the antigen binding ability of non-transduced (NTD) control T cells, and D1-D4 CAR-T cells. T cells were incubated with human IgG Fc-tagged CLL-1 recombinant protein and His-tagged CD33 recombinant protein. After washing, cells were further stained with secondary PE-conjugated anti-His antibody (Cat # 362603, BioLegend) and Alexa Fluor® 647 AffiniPure™ F(ab')2 Fragment Goat Anti-Human IgG, Fey fragment specific antibody (Code 109-606-008, Jackson ImmunoResearch Inc.). (C) Flow cytometry assays confirmed single and double knockout of CLL-1 / CD33 in AML single clones generated from HL-60-Luc2 cells and U-937-GFP-Luc2 cells byCRISPR-Cas9 gene editing technology. (D) IFNy levels from the co-culture of indicated T cells and AML cells. T cells (50K total T cells / well) were co-cultured with wild type (WT) or CLL-1 / CD33 single / double knockout HL-60 or U-937 cells (50K cells / well) overnight. Cytokine levels in the co-culture supernatants were quantitated by cytokine bead array (CBA, BD Biosciences).

[0079] Figures 3A-3F. CLL-l / CD33-specific dual-CAR T cells with two different sets of hinge / TM domains display significantly reduced leakage to CD33-single positive targets. (A) Schematic diagrams show the structures of dual-CARs with different sets of hinge (H) / TM domains after P2A (H / TM domains from CD8 for DC-3; H / TM domains from CD28 for DC-3-28HTM). The sequences of anti-CLL-1 and anti-CD33 single variable domain on a heavy chain (VHH) antibodies (CLL-11 and CD33.16) were used. Secondary-generation mono-specific CARs (CLL-11 and CD33.7) were used as controls. (B) CAR expressions on control and dual-CAR T cells were determined by antigen binding assays. (C-D) IFNy production from control and dual-CAR T cells after overnight co-culture with HL-60 cells (C) or U-937 cells (D) was quantitated by CBA. Mono-specific CARs were used to confirm CLL-1 expression (CLL-11) or CD33 expression (CD33.7) on both single KO and dKO AML cells. (E-F) Luciferase killing assays showed that DC-3-28HTM dual-CAR T cells are more potent in killing CLL-l / CD33-double positive AML cells in comparison to their killing of CD33-single positive AML cells. HL-60-Luc2 cells (E) and U-937-GFP-Luc2 cells (F) were used as the target cells. RLU, relative light unit.

[0080] Figures 4A-4F. Comparisons of CLL-l / CD33-specific dual-CAR T cells with different sets of hinge / TM domains. (A) Schematic diagrams show the structures of dual-CARs. "-28HTM", hinge / TM domains after P2A are from CD28; "-8-28", the signal peptide / hinge / TM sets before and after P2A are from CD8 and CD28, respectively; "-28-8", the signal peptide / hinge / TM sets before and after P2A are from CD28 and CD8, respectively. (B) CAR expressions on control T and dual-CAR T cells were determined by antigen binding assays. (C-D) IFNy production from control and dual-CAR T cells after overnight co-culture with HL-60 cells (C) or U-937 cells (D) was quantitated by CBA. (E-F) CLL-l / CD33-specific “-28-8” framework dual-CAR T cells (DC-3-28-8) differentially kill CD33 single antigen-expressing and CLL-1 / CD33 double antigensexpressing HL-60-Luc2 cells (E) and U-937-GFP-Luc2 cells (F).

[0081] Figures 5A-5F. Characterization of candidate anti-CLL-1 VHHs that were discovered through camelid immunization and library screening. (A) Schematic diagram shows the structure of anti-CLL-1 VHH and hlgGl fusion proteins used for characterization. (B) EC50 valuesof 12 anti-CLL-1 VHHs, including CLL-A01 to CLL-A12, were determined by enzyme-linked immunosorbent assay (ELISA). Human IgGl-Fc linked VHHs were incubated with plate-bound His-tagged recombinant CLL-1 protein. Horseradish peroxidase (HRP)-conjugated anti-human IgG-Fc 2udantibody was applied and OD450nm values were obtained to calculate EC50. (C) The affinity (KD, the equilibrium dissociation constant) of anti-CLL-1 VHHs was determined by surface plasmon resonance (SPR). (D-F) The percentages of VHH-bound cells after incubating candidate VHHs with CLL-1 -overexpressing A549 cells (D), HL-60 cells (E) and U-937 cells (F) were summarized. FITC-2nd, FITC-conjugated anti-hlgG Fc 2ndantibody only.

[0082] Figures 6A-6F. Comparisons of IFNy production from CLL-1 mono-specific CLL-Ax CAR-T cells after co-culture with AML cells. (A) Schematic diagrams show the structures of CLL-Ax CARs. (B) CAR expressions on control T and CLL-Ax CAR-T cells were determined by antigen binding assays. (C-F) IFNy production from control T and CLL-Ax CAR-T cells after overnight incubation with wild type HL-60 cells (C), CLL-1 single knockout HL-60 cells (D), wild type U-937 cells (E), and CLL-1 single knockout U-937 cells (F) was quantitated by CBA. Equal number of CAR-positive T cells (50K CAR+cells / well) were used for comparisons. ScFv-based CAR (CLL-06) and VHH-based CARs (CLL-10 and CLL-11) were used as benchmarks.

[0083] Figures 7A-7B. CLL-1 mono-specific CLL-Ax CAR-T cells show excellent potency in killing AML cells. (A-B) Luciferase killing assays demonstrate that the selected CLL-Ax CAR-T cells mediate comparable antitumor responses in killing HL-60-Luc2 cells (A) and U-937-GFP-Luc2 cells (B) as the benchmarks: scFv-based CAR-T (CLL-06) and VHH-based CAR-T (CLL-10) cells.

[0084] Figures 8A-8F. Characterization of candidate anti-CD33 VHHs that were discovered through camelid immunization and library screening. (A) Schematic diagram shows the structure of anti-CD33 VHH and hlgGl fusion proteins used for characterization. (B) EC50 values of 11 anti-CD33 VHHs. including CD33-B01 to CD33-B11, were determined by ELISA. Human IgGl-Fc linked VHHs were incubated with plate-bound His-tagged recombinant CD33 protein. HRP-conjugated anti-human IgG-Fc 2ndantibody was applied and OD450nm values were obtained to calculate EC50. (C) The affinity (KD) of anti-CD33 VHHs was determined by surface SPR. (D-F) The percentages of VHH-bound cells after incubating candidate VHHs with CD33-0 verexpressing A549 cells (D), HL-60 cells (E) and U-937 cells (F) were summarized. FITC, FITC-conjugated anti-IgG Fc 2udantibody only.Figures 9A-9F. Comparisons of IFNy production from CD33 mono-specific CD33-Bx CAR-T cells after co-culture with AML cells. (A) Schematic diagrams show the structures of CD33-Bx CARs. (B) CAR expressions on control and CD33-Bx CAR-T cells were determined by antigen binding assays. (C-F) IFNy production from control T and CD33-Bx CAR-T cells after overnight incubation with wild type HL-60 cells (C), CD33 single knockout HL-60 cells (D), wild type U-937 cells (E), and CD33 single knockout U-937 cells (F) was quantitated by CBA. Equal number of CAR-positive T cells (50K CAR+cells / well) were used for comparisons. ScFv-based CAR (CD33.7) and VHH-based CARs (CD33.16 and CD33.17) were used as benchmarks.

[0085] Figures 10A-10D. Co-culture experiments demonstrate that all the tested VHH combinations are favorable for CLL-1 / CD33 dual targeting. (A) Schematic diagrams show the structures of AxBy-28HTM dual-CARs. Selected CLL-Ax VHH (including A04, A07, and A09) and CD33-By VHH (including B04, B06, and Bll) candidates were used. (B) CAR expressions on control T (NTD), CLL-1 mono CAR-T (CLL-sCAR; CLL-Ax CAR-T), CD33 mono CAR-T (CD33-SCAR; CD33-By CAR-T). and CLL-1 / CD33 dual-CAR T (AxBy-28HTM) cells were determined by antigen binding assays. Representative flow profile shows the binding of T cells to human IgG Fc-tagged CLL-1 recombinant protein and His-tagged CD33 recombinant protein. (C-D) IFNy production from the indicated T cells after overnight incubation with HL-60 cells (C) or U-937 cells (D) was quantitated by CBA. DC-3-28HTM dual-CAR was used as a benchmark.

[0086] Figures 11A-11D. Comparison of "-28HTM" and "-28-8" framework dual-CARs with CLL-A09 and CD33-B06 or CD33-B11 VHH combinations. (A) Schematic diagrams show the structures of A09By-28HTM and A09By-28-8 dual-CARs. (B) CAR expressions on control T (NTD). CLL-A09 CAR-T, CD33-By CAR-T. A09By-28HTM dual-CAR T and A09By-28-8 dual-CAR T cells were determined by antigen binding assays. (C-D) IFNy production from the indicated T cells after overnight incubation with HL-60 cells (C) or U-937 cells (D) was quantitated by CBA.

[0087] Figures 12A-12D. A07B11-28HTM dual-CAR T cells show differential potency against CD33 single antigen- and CLL-1 / CD33 double antigens-expressing AML cells. (A) Schematic diagrams show the structures of A07B11-28HTM dual-CAR. (B) CAR expressions on control T (NTD), CLL-A07 CAR-T, CD33-B11 CAR-T, and A07B11-28HTM dual-CAR T cells were determined by antigen binding assays. (C) IFNy production from the indicated T cells after overnight incubation with HL-60-Luc2 cells (upper panel) or U-937-GFP-Luc2 cells (lower panel)bearing the indicated genotypes was quantitated by CBA. A07B11-28HTM dual-CAR T cells showed differential IFNy release upon coculture with CLL-1 / CD33 single-positive versus doublepositive AML cells. (D) Luciferase killing assays demonstrate that A07B11-28HTM dual-CAR T cells exhibited differential killing ability against CLL-l / CD33-single antigen-expressing and CLL-1 / CD33 double antigen-expressing HL-60-Luc2 cells (upper panel) and U-937-GFP-Luc2 cells (lower panel). Target AML cells at the indicated genotypes were incubated with effector A07B11-28HTM dual-CAR T cells overnight at various effector-to-target (E : T) ratios. The percentages of relative light unit (RLU) values in groups with both T cells and target cells to the corresponding target-only groups (E : T=0 : 1) were summarized.

[0088] Figures 13A-13D. Codon-optimized A07B11-28HTM (V3.2) dual-CAR T cells show differential potency against CD33 single antigen- and CLL-1 / CD33 double antigensexpressing target cells. (A) A07B11-28HTM with codon optimization (V3.2) did not result in major changes in CAR expression. CLL-1 CAR and CD33 CCR expressions in control (NTD) and dual-CAR T cells were determined by antigen binding assays. (B) IFNy production from the indicated T cells after overnight incubation with HL-60 cells (upper panel) or U-937 cells (lower panel) was quantitated by CBA. (C) Luciferase killing assays demonstrate that A07B11-28HTM (left) and A07B11-28HTM-V3.2 (right) dual-CAR T cells differentially killed CD33-single antigen- and CLL-1 / CD33 double antigens-expressing HL-60-Luc2 cells. (D) A07B11-28HTM and A07B11-28HTM-V3.2 showed similar differential potency against CD33-single antigen- and CLL-1 / CD33 double antigens-overexpressing (OE) A549 cells. IFNy production from T cells after overnight incubation with WT (CLL-1 CD33 ), CD33 OE (CLL-1 CD33+), CLL-1 OE (CLL-UCD33 ), and double OE (dOE; CLL-1+CD33+) A549 cells was quantitated by CBA. The cytolytic activity of T cells against these A549 cells after 24 hours’ incubation was evaluated using the xCELLigence real-time cell analysis (RTCA) system.

[0089] Figure 14 shows dual-targeting of CLL-1 and CD33 increases T cell sensitivity to low-density CLL-1. MOLM-13 cells are CLL-1LOW / CD33+. Luciferase killing assay: RLU refers to relative light unit. CLL-11, 2ndgeneration anti-CLL-1 mono-specific CAR; CD33.16, 2ndgeneration anti-CD33 mono-specific CAR; DC-3-28HTM and DC-3-28-8, anti-CLL-1 and anti-CD33 dual-CAR.

[0090] Figure 15 is a scheme showing the anti-CLL-1 VHH and anti-CD33 VHH generation process.DETAILED DESCRIPTION

[0091] The present disclosure provides CLL-1 / CD33 dual-targeting chimeric antigen receptors (CARs). The CAR may comprise an anti-CLL-1 antigen-binding region and an anti-CD33 antigenbinding region. The CAR may comprise an anti-CLL-1 VHH and an anti-CD33 VHH. The dualtargeting CAR may comprise two portions: one having a first VHH, a first hinge region, a first transmembrane domain, and a cytoplasmic signaling domain; the other having a second VHH, a second hinge region, a second transmembrane domain, and a co- stimulatory region. The present CARs can be used to treat cancer, such as acute myeloid leukemia (AML).

[0092] Dual-targeting CAR-T cells may show substantial activity only when both targets are present on the tumor and thus possess improved safety. The dual-CAR approach minimizes parallel reactivity against normal tissues bearing a single antigen.

[0093] CLL-1 (C-type lectin-like molecule 1), also known as CLEC12A (C-type lectin domain family 12 member A) or CD37L is expressed on leukemic blasts in a large proportion of AML patients and is also expressed on leukemic stem cells (LSCs). In normal hematopoiesis, CLL-1 expression emerges at the level of the common myeloid progenitor, extending towards the mature myeloid lineage. CLL-1 is a stable marker for detection of measurable residual disease in AML. CLL-1, as used herein, may refer to any native, mature CLL-1 that results from processing of a CLL-1 precursor in a cell. The term includes CLL-1 from any vertebrate, including mammals such as primates (e.g., humans, cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring variants of CLL-1, such as splice variants or allelic variants. A non-limiting exemplary mature human CLL-1 amino acid sequence is shown, e.g., in UniProt Accession No. Q5QGZ9.

[0094] CD33 (myeloid cell surface antigen CD33), also known as Siglec-3 (sialic acid-binding Ig-like lectin 3) or gp67, is an antigen marker that is present on AML blasts in approximately 90% patients, making it a potent distinguisher of AML blasts. However, CD33 is expressed on hematopoietic stem cells and hepatocytes, making them susceptible for CD33-targeted immunotherapies (such as Gemtuzumab ozogamicin (GO), a CD33-directed antibody-drug conjugate). CD33, as used herein, may refer to any native, mature CD33 that results from processing of a CD33 precursor in a cell. The term includes CD33 from any vertebrate, including mammals such as primates (e.g., humans, cynomolgus or rhesus monkeys) and rodents (e.g.,mice and rats). The term also includes naturally occurring variants of CD33, such as splice variants or allelic variants. A non-limiting exemplary mature human CD33 amino acid sequence is shown, e.g., in UniProt Accession No. P20138.

[0095] CLL-1 and CD33 are co-expressed on AML blasts and LSCs.

[0096] The present disclosure provides for a nucleic acid, or a system, comprising (i) a first polynucleotide encoding a first polypeptide, and (ii) a second polynucleotide encoding a second polypeptide. The first polypeptide may comprise an anti-CLL-1 antigen-binding region comprising a first VHH (i.e., an anti-CLL-1 VHH). The second polypeptide may comprise an anti-CD33 antigen-binding region comprising a second VHH (i.e., an anti-CD33 VHH).

[0097] The first polynucleotide and the second polynucleotide may be on a single nucleic acid molecule, or on separate nucleic acid molecules.

[0098] The present disclosure provides for a recombinant polypeptide comprising an anti-CLL-1 VHH. The recombinant polypeptide may be a chimeric antigen receptor (CAR) or an antibody.

[0099] The present disclosure also provides for a recombinant polypeptide comprising an anti-CD33 VHH. The recombinant polypeptide may be a chimeric antigen receptor (CAR) or an antibody.

[0100] The (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise one, two, or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, respectively; (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively; (4) SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, respectively; (5) SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, respectively; (6) SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, respectively; (7) SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90. respectively; (8) SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, respectively; (9) SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, respectively; (10) SEQ IDNO: 166, SEQ ID NO: 168, SEQ ID NO: 170, respectively; (11) SEQ ID NO: 182, SEQ ID NO:184, SEQ ID NO:186, respectively; or (12) SEQ ID NO:198, SEQ ID N0:200, SEQ ID NO:202, respectively.

[0101] The (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise one, two, or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%. at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, respectively; or (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154. respectively.

[0102] The (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise one, two, or three three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively; (4) SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, respectively; (5) SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, respectively; (6) SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, respectively; (7) SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, respectively; (8) SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, respectively; (9) SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, respectively; (10) SEQ ID NO:390. SEQ ID NO:392, SEQ ID NO:394, respectively; or (11) SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, respectively.The (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise one, two, or three three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%. at least or about 88%, at least or about 89%, at least or about 90%. at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%. or about 100%, identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; or (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively.

[0103] The (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%. at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%. at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, SEQ ID NO: 147. SEQ ID NO:19, SEQ ID NO:35, SEQ ID NO:51, SEQ ID NO:83, SEQ ID NO:99, SEQ ID NO:131, SEQ ID NO: 163, SEQ ID NO: 179, or SEQ ID NO: 195.

[0104] The (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%. at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, or SEQ ID NO: 147.

[0105] The (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at leastor about 75%, at least or about 80%, at least or about 81 %, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, SEQ ID NO:419, SEQ ID NO:259, SEQ ID NO:275, SEQ ID NO: 291, SEQ ID NO:323, SEQ ID NO:355, SEQ ID NO:371, SEQ ID NO:387, or SEQ ID NO:403.

[0106] The (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%. at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, or SEQ ID NO:419.

[0107] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise one, two, or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%. at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively;

[0108] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise one, two, or three three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least orabout 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively.

[0109] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise one, two, or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%. at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively;

[0110] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise one, two, or three three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively.

[0111] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise one, two, or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%. at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively;

[0112] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise one, two, or three three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively.

[0113] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise one, two, or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%. at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, respectively;

[0114] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise one, two. or three three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%. at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, atleast or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively.

[0115] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise one, two, or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, respectively;

[0116] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise one, two, or three three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%. at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively.

[0117] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise one, two, or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least orabout 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, respectively;

[0118] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise one, two, or three three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3. having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%. at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively.

[0119] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise one, two, or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively;

[0120] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise one, two, or three three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%,identical to the amino acid sequences set forth in SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively.

[0121] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise one, two, or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%. at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO: 150, SEQ ID NO:152, SEQ ID NO:154, respectively;

[0122] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise one, two, or three three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively.

[0123] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise one, two, or three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3. having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%. at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO: 150, SEQ IDNO: 152, SEQ ID NO: 154, respectively;

[0124] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise one, two, or three three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%. at least or about 84%, at least or about 85%, at least or about 86%. at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%. at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequences set forth in SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively.

[0125] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%. at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%. at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 67;

[0126] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%. at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%. at least or about 92%. at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:307.

[0127] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%. at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, atleast or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 67;

[0128] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:339.

[0129] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 67;

[0130] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%. at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:419.

[0131] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%. at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 115;

[0132] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:307.

[0133] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 115;

[0134] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:339.

[0135] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise an amino acid sequence about 80% to about 100%, at least orabout 70%, at least or about 75%, at least or about 80%, at least or about 81 %, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%. at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 115:

[0136] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%. at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:419.

[0137] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%. at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 147:

[0138] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%. at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%. at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:307.

[0139] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or theanti-CLL-1 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%. at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 147;

[0140] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:339.

[0141] In certain embodiments, the (first) VHH of the anti-CLL-1 antigen-binding region (or the anti-CLL-1 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 147;

[0142] the (second) VHH of the anti-CD33 antigen-binding region (or the anti-CD33 VHH) may comprise an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:419.The recombinant polypeptide may be an anti-CLL-1 CAR, comprising an amino acid sequence about 80% to about 100%, at least or about 70%. at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%. at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%. at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO: 225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231. SEQ ID NO:233. SEQ ID NO: 235. SEQ ID NO:237. SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO: 245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, or SEQ ID NO:257.

[0143] The recombinant polypeptide may be an anti-CD33 CAR, comprising an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:435, SEQ ID NO:437, SEQ ID NO:439, SEQ ID NO:441, SEQ ID NO:443, SEQ ID NO:445. SEQ ID NO:447. SEQ ID NO:449, SEQ ID NO:451, SEQ ID NO:453, SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459, SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465, SEQ ID NO:467, SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID NO:475, or SEQ ID NO:477.

[0144] In certain embodiments, the first polypeptide is a first chimeric antigen receptor (CAR) component, and the second polypeptide is a second CAR component.

[0145] The CAR or CAR component (e.g., the first CAR component, or the second CAR component) may further comprise one or more of the following: (a) a signal peptide or SP (e.g., a first signal peptide, or a second signal peptide), or a leader sequence, (b) a hinge region (e.g., afirst hinge region, or a second hinge region), (c) a transmembrane domain (e.g., a first transmembrane domain, or a second transmembrane domain), (d) a co-stimulatory region (e.g., a first co-stimulatory region, or a second co-stimulatory region), and (e) a cytoplasmic signaling domain (e.g., a first cytoplasmic signaling domain, or a second cytoplasmic signaling domain).

[0146] The first polypeptide may further comprise a first hinge region, a first transmembrane domain, or a combination thereof. The second polypeptide may further comprise a second hinge region, a second transmembrane domain, or a combination thereof. The first hinge region may be different from, or the same as, the second hinge region. The first transmembrane domain may be different from, or the same as, the second transmembrane domain.

[0147] The first polypeptide may further comprise a first cytoplasmic signaling domain. The second polypeptide may further comprise a second co-stimulatory region.

[0148] The first polypeptide may further comprise a first co-stimulatory region. The second polypeptide may further comprise a second cytoplasmic signaling domain.

[0149] The first polynucleotide may be located at the 5’ end of the second polynucleotide. The second polynucleotide may be located at the 5’ end of the first polynucleotide.

[0150] In certain embodiments, the first polypeptide comprises from N-terminus to C-terminus: the first VHH, a first hinge region, a first transmembrane domain, and a first cytoplasmic signaling domain.

[0151] In certain embodiments, the second polypeptide comprises from N-terminus to C-terminus: the second VHH, a second hinge region, a second transmembrane domain, and a second co-stimulatory region.

[0152] The present CAR or CAR component (e.g., the first CAR component, or the second CAR component) may comprise, from N-terminus to C-terminus, a signal peptide, a VHH, a hinge region, a transmembrane domain, a co-stimulatory region, and a cytoplasmic signaling domain.

[0153] The present CAR or CAR component (e.g., the first CAR component, or the second CAR component) may comprise, from N-terminus to C-terminus, a signal peptide, a VHH, a hinge region, a transmembrane domain, and a co-stimulatory region.

[0154] The present CAR or CAR component (e.g., the first CAR component, or the second CAR component) may comprise, from N-terminus to C-terminus, a signal peptide, a VHH, a hinge region, a transmembrane domain, and a cytoplasmic signaling domain.

[0155] The first polypeptide and the second polynucleotide may be linked by a linker. In certainembodiments, the linker is a cleavable linker or non-cleavable linker.

[0156] The first polypeptide may further comprise a first signal peptide. The second polypeptide may further comprise a second signal peptide. The first signal peptide may be the same as, or different from, the second signal peptide.

[0157] In certain embodiments, the first polynucleotide comprises a nucleotide sequence about 80% to about 100%. at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the nucleotide sequence set forth in SEQ ID NO: 482, SEQ ID NO:484, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:502, SEQ ID NO:504, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:522, SEQ ID NO:524, SEQ ID NO:532. SEQ ID NO:534, SEQ ID NO:542, SEQ ID NO:544. SEQ ID NO:552, SEQ ID NO:554, SEQ ID NO:562, SEQ ID NO:564, SEQ ID NO:572, or SEQ ID NO:574.

[0158] In certain embodiments, the second polynucleotide comprises a nucleotide sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%. or about 100%, identical to the nucleotide sequence set forth in SEQ ID NO:486, SEQ ID NO:488, SEQ ID NO:496, SEQ ID NO:498, SEQ ID NO:506, SEQ ID NO:508, SEQ ID NO:516, SEQ ID NO:518, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:536, SEQ ID NO:538, SEQ ID NO:546. SEQ ID NO:548. SEQ ID NO:556, SEQ ID NO:558, SEQ ID NO:566, SEQ ID NO:568, SEQ ID NO:576, or SEQ ID NO:578.

[0159] In certain embodiments, the first polypeptide comprises an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:481, SEQ ID NO:483, SEQ ID NO:491. SEQ ID NO:493, SEQ ID NO:501, SEQ ID NO:503, SEQ ID NO:511, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:523, SEQ ID NO:531, SEQ ID NO:533, SEQ ID NO:541, SEQ ID NO:543, SEQ ID NO:551, SEQ ID NO:553, SEQ ID NO:561, SEQ ID NO:563, SEQ ID NO:571. or SEQ ID NO:573.

[0160] In certain embodiments, the second polypeptide comprises an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:485, SEQ ID NO:487. SEQ ID NO:495, SEQ ID NO:497, SEQ ID NO:505, SEQ ID NO:507, SEQ ID NO:515, SEQ ID NO:517, SEQ ID NO:525, SEQ ID NO:527, SEQ ID NO:535, SEQ ID NO:537, SEQ ID NO:545, SEQ ID NO:547, SEQ ID NO:555, SEQ ID NO:557, SEQ ID NO:565, SEQ ID NO:567, SEQ ID NO:575, or SEQ ID NO:577.

[0161] In certain embodiments, in the polypeptide encoded by the present nucleic acid, a cytoplasmic signaling domain is located at the N-terminus of a co-stimulatory region. In certain embodiments, a co-stimulatory region is located at the N-terminus of a cytoplasmic signaling domain.

[0162] In certain embodiments, in the polypeptide encoded by the present nucleic acid, the anti-CLL-1 antigen-binding region is located at the N-terminus of the anti-CD33 antigen-binding region. In certain embodiments, the anti-CD33 antigen-binding region is located at the N-terminus of the anti-CLL- 1 antigen-binding region.

[0163] In certain embodiments, the nucleic acid comprises a nucleotide sequence about 80% to about 100%, at least or about 70%. at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%. at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, atleast or about 99%, or about 100%, identical to the nucleotide sequence set forth in SEQ ID NO:480, SEQ ID NO:490, SEQ ID NO:500, SEQ ID NO:510, SEQ ID NO:520, SEQ ID NO:530, SEQ ID NO:540, SEQ ID NO:550. SEQ ID NO:560. or SEQ ID NO:570.

[0164] The polypeptide encoded by the present nucleic acid may comprise, from N-terminus to C-terminus, a first signal peptide, a first VHH, a first hinge region, a first transmembrane domain, a first cytoplasmic signaling domain, a linker, a second signal peptide, a second VHH, a second hinge region, a second transmembrane domain, and a second co-stimulatory region.

[0165] The polypeptide encoded by the present nucleic acid may comprise, from N-terminus to C-terminus, a first VHH, a first hinge region, a first transmembrane domain, a first cytoplasmic signaling domain, a linker, a second VHH, a second hinge region, a second transmembrane domain, and a second co-stimulatory region.

[0166] The polypeptide encoded by the present nucleic acid may comprise, from N-terminus to C-terminus, a first signal peptide, a first VHH, a first hinge region, a first transmembrane domain, a first co-stimulatory region, a linker, a second signal peptide, a second VHH, a second hinge region, a second transmembrane domain, and a second cytoplasmic signaling domain.

[0167] The polypeptide encoded by the present nucleic acid may comprise, from N-terminus to C-terminus, a first VHH, a first hinge region, a first transmembrane domain, a first co-stimulatory region, a linker, a second VHH, a second hinge region, a second transmembrane domain, and a second cytoplasmic signaling domain.

[0168] The polypeptide encoded by the present nucleic acid may comprise, from N-terminus to C-terminus, a second signal peptide, a second VHH, a second hinge region, a second transmembrane domain, a second co-stimulatory region, a linker, a first signal peptide, a first VHH, a first hinge region, a first transmembrane domain, and a first cytoplasmic signaling domain.

[0169] The polypeptide encoded by the present nucleic acid may comprise, from N-terminus to C-terminus. a second VHH, a second hinge region, a second transmembrane domain, a second co-stimulatory region, a linker, a first VHH, a first hinge region, a first transmembrane domain, and a first cytoplasmic signaling domain.

[0170] The polypeptide encoded by the present nucleic acid may comprise, from N-terminus to C-terminus, a second signal peptide, a second VHH, a second hinge region, a second transmembrane domain, a second cytoplasmic signaling domain, a linker, a first signal peptide, a first VHH, a first hinge region, a first transmembrane domain, and a first co-stimulatory region.The polypeptide encoded by the present nucleic acid may comprise, from N-terminus to C-terminus, a second VHH, a second hinge region, a second transmembrane domain, and a second cytoplasmic signaling domain, a linker, a first VHH, a first hinge region, a first transmembrane domain, and a first co-stimulatory region.

[0171] In one embodiment, the first CAR (component) comprises, from N-terminus to C-terminus, (i) an anti-CLL-1 antigen-binding region comprising a first VHH (an anti-CLL-1 VHH), (ii) a first hinge region of (or derived from) CD8, (iii) a first transmembrane domain of (or derived from) CD8, and (iv) a first cytoplasmic signaling domain of (or derived from) CD3zeta. In one embodiment, the second CAR (component) comprises, from N-terminus to C-terminus, (i) an anti-CD33 antigen-binding region comprising a second VHH (an anti-CD33 VHH), (ii) a second hinge region of (or derived from) CD28, (iii) a second transmembrane domain of (or derived from) CD28, and (iv) a second co-stimulatory region of (or derived from) 4- IBB.

[0172] When the first CAR (component) and the second CAR (component) are expressed as a single polypeptide before cleavage, the first CAR (component) may be at the N-terminus of the second CAR (component), and the first CAR (component) and the second CAR (component) are linked by a cleavable linker such as a 2 A peptide (e.g., P2A, E2A, T2A, or F2A).

[0173] In one embodiment, the first CAR (component) comprises, from N-terminus to C-terminus, (i) an anti-CLL-1 antigen-binding region comprising a first VHH (an anti-CLL-1 VHH), (ii) a first hinge region of (or derived from) CD28, (iii) a first transmembrane domain of (or derived from) CD28, and (iv) a first cytoplasmic signaling domain of (or derived from) CD3zeta. In one embodiment, the second CAR (component) comprises, from N-terminus to C-terminus, (i) an anti-CD33 antigen-binding region comprising a second VHH (an anti-CD33 VHH), (ii) a second hinge region of (or derived from) CD8, (iii) a second transmembrane domain of (or derived from) CD8, and (iv) a second co-stimulatory region of (or derived from) 4- IBB.

[0174] When the first CAR (component) and the second CAR (component) may be expressed as a single polypeptide before cleavage, the first CAR (component) is at the N-terminus of the second CAR (component), and the first CAR (component) and the second CAR (component) are linked by a cleavable linker such as a 2A peptide (e.g., P2A, E2A, T2A, or F2A).

[0175] In certain embodiments, the nucleic acid encodes a polypeptide comprising an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about80%, at least or about 81 %, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%. at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:479, SEQ ID NO:489, SEQ ID NO:499, SEQ ID NO:509, SEQ ID NO:519, SEQ ID NO:529, SEQ ID NO:539, SEQ ID NO:549, SEQ ID NO:559, or SEQ ID NO:569.

[0176] The signal peptide (e.g., the first signal peptide, or the second signal peptide) may comprise a signal peptide of (or may be derived from) CD8, CD28, GM-CSF, CD4, CD137, or combinations thereof. In one embodiment, the signal peptide is a signal peptide of (or is derived from) CD8 (e.g., SEQ ID NO:1). In one embodiment, the first signal peptide is a signal peptide of (or is derived from) CD8. In one embodiment, the second signal peptide is a signal peptide of (or is derived from) CD8. In one embodiment, the signal peptide is a signal peptide of (or is derived from) CD28 (e.g., SEQ ID NO: 13). In one embodiment, the first signal peptide is a signal peptide of (or is derived from) CD28. In one embodiment, the second signal peptide is a signal peptide of (or is derived from) CD28.

[0177] In one embodiment, the signal peptide (e.g., the first signal peptide, or the second signal peptide) comprises an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO: 13.

[0178] The hinge region (e.g., the first hinge region, or the second hinge region) may comprise a hinge region of (or may be derived from) IgG4, CD8, CD28, CD137. or combinations thereof, wildtype or mutants.

[0179] In one embodiment, the hinge region (e.g., the first hinge region, or the second hinge region) is a hinge region of (or is derived from) CD8 (e.g., SEQ ID NO:3) or CD28 (e.g., SEQ ID NO: 15). In one embodiment, the first hinge region is a hinge region of (or is derived from) CD8.In one embodiment, the second hinge region is a hinge region of (or is derived from) CD28. In one embodiment, the hinge region (e.g., the first hinge region, or the second hinge region) comprises an amino acid sequence about 80% to about 100%. at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%. at least or about 89%, at least or about 90%, at least or about 91%. at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%. at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO: 15.

[0180] In one embodiment, the first hinge region comprises an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%. at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%. at least or about 91%, at least or about 92%, at least or about 93%. at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:3.

[0181] In one embodiment, the second hinge region comprises an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:15.

[0182] The transmembrane domain (e.g.. the first transmembrane domain, or the second transmembrane domain) may comprise a transmembrane domain of (or may be derived from) CD8, CD28, CD3s, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof.

[0183] In one embodiment, the transmembrane domain (e.g., the first transmembrane domain, or the second transmembrane domain) is a transmembrane domain of (or is derived from) CD8 (e.g., SEQ ID NO:5) or CD28 (e.g., SEQ ID NO: 17). In one embodiment, the first transmembranedomain is a transmembrane domain of (or is derived from) CD8. In one embodiment, the second transmembrane domain is a transmembrane domain of (or is derived from) CD28.

[0184] In one embodiment, the transmembrane domain (e.g., the first transmembrane domain, or the second transmembrane domain) comprises an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:17.

[0185] In one embodiment, the first transmembrane domain comprises an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:5.

[0186] In one embodiment, the second transmembrane domain comprises an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 17.

[0187] The co-stimulatory region (e.g., the first co- stimulatory region, or the second costimulatory region) may comprise a co-stimulatory region of (or may be derived from) 4- IBB (CD137), CD28, 0X40, CD2, CD7, CD27, CD30, CD40, CD70. CD134, PD1, DaplO, CDS, ICAM-1, LFA-1 (CDlla / CD18), ICOS (CD278), NKG2D, GITR, TLR2, TIM1, HVEM, orcombinations thereof.

[0188] In one embodiment, the co-stimulatory region (e.g., the first co- stimulatory region, or the second co-stimulatory region) is a co-stimulatory region of (or is derived from) 4- IBB (e.g., SEQ ID NO:7).

[0189] In one embodiment, the co-stimulatory region (e.g., the first co-stimulatory region, or the second co-stimulatory region) comprises an amino acid sequence about 80% to about 100%. at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%. at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:7.

[0190] The cytoplasmic signaling domain (e.g., the first cytoplasmic signaling domain, or the second cytoplasmic signaling domain) may comprise a cytoplasmic signaling domain of (or may be derived from) CD3i^ (e.g., SEQ ID NO:9).

[0191] In one embodiment, the cytoplasmic signaling domain (e.g., the first cytoplasmic signaling domain, or the second cytoplasmic signaling domain) comprises an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%. at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO:9.

[0192] In certain embodiments, the first polypeptide and the second polypeptide are expressed as a single polypeptide separated by a linker. In certain embodiments, the first polynucleotide and the second polynucleotide are separated by an oligonucleotide encoding a linker. The linker may be cleavable or non-cleavable. In one embodiment, the linker is a 2A peptide, such as P2A E2A, T2A, or F2A.

[0193] In one embodiment, the linker comprises an amino acid sequence about 80% to about 100%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 81%. at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%. at least or about 96%, at least or about 97%, at least or about 98%. at least or about 99%, or about 100%, identical to the amino acid sequence set forth in SEQ ID NO: 11.

[0194] In certain embodiments, the present nucleic acid encodes a bispecific antibody, where the first polypeptide is a first antibody fragment, and the second polypeptide is a second antibody fragment.

[0195] In certain embodiments, the present recombinant polypeptide is an antibody.

[0196] The present recombinant polypeptide, or the first or second polypeptide, may comprise at least one VHH domain, such as one, two, three, four or more VHH domains, where each VHH domain may be the same or different.

[0197] In certain embodiments, the present recombinant polypeptide, or the first or second polypeptide, comprises an Fc region, such as a human Fc region, including a human IgG Fc region. Further, in some such embodiments, the polypeptide may form a dimer. In certain embodiments, the polypeptide may include VHHl-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be the same or different. An Fc region may refer to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, an Fc region comprises a hinge, CH2, and CH3. An Fc region may be of any antibody heavy chain constant region isotype. In some embodiments, an Fc region is an Fc region of IgGl, IgG2, IgG3, or IgG4.

[0198] In some embodiments, an antibody is a multi- specific antibody. For example, it comprises a plurality of variable domain sequences (e.g., VHHs), wherein a first variable domain sequence has binding specificity for a first epitope and a second variable domain sequence has binding specificity for a second epitope.

[0199] In some embodiments, a multi- specific antibody molecule is a bispecific or trispecific antibody molecule. A bispecific antibody has specificity for two antigens. A bispecific antibody molecule is characterized by a first variable domain sequence which has binding specificity for a first epitope (e.g., CLL-1) and a second variable domain sequence that has binding specificity for a second epitope (e.g., CD33). A bispecific antibody refers to a molecule that combines the antigen binding sites of two antibodies within a single molecule. Thus, a bispecific antibody can bind two different antigens simultaneously or sequentially.The present disclosure provides for one or more vectors (or at least one vector) comprising the present nucleic acid, or the present system. The vector may comprise DNA or RNA. The vector may be a plasmid, virus vector, transposon, or combinations thereof. The vector may comprise a DNA virus or a retroviral vector. The vector may be a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or combinations thereof. In one embodiment, the vector is a lentiviral vector.

[0200] The present disclosure provides for an immune cell expressing or comprising the present CAR, the present antibody, the present recombinant polypeptide, or the first and second polypeptides.

[0201] The present disclosure provides an immune cell, comprising the present nucleic acid, the present system, or the vector.

[0202] The immune cell may be a T cell or a natural killer (NK) cell.

[0203] The immune cell may be an isolated cell. The cell may be a genetically engineered cell. The cell may be a mammalian cell. In one embodiment, the cell is a CAR-T cell and / or a CAR-NK cell.

[0204] The present disclosure also provides for a pharmaceutical composition, comprising the present CAR, the present antibody, the present immune cell, the present nucleic acid, the present system, the present vector, the present recombinant polypeptide, or the first and second polypeptides. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may be a liquid preparation.

[0205] The pharmaceutical composition may comprise the present immune cells at a concentration ranging from about IxlO3cells / mLto about IxlO8cells / mL, or from about IxlO4cells / mL to about IxlO7cells / mL.

[0206] The present disclosure also provides for a method of treating cancer. The method may comprise administering the present immune cell or present pharmaceutical composition to a subject in need thereof.

[0207] The immune cell may be allogeneic or autologous.

[0208] The cancer may be a hematologic cancer. The cancer may be a lymphoma or leukemia. The cancer may be Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, or multiple myeloma. Thecancer may be a B-cell malignancy. The cancer may be acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), or combinations thereof.

[0209] The present disclosure provides a method for preparing an immune cell expressing the chimeric antigen receptor (e.g., a CAR-T cell), where the method comprises: transducing the present nucleic acid or the present vector into an immune cell (e.g., a T cell or NK cell), thereby obtaining the immune cell expressing the chimeric antigen receptor (e.g., the CAR-T cell).

[0210] The antigen-binding region may refer to a portion of an antibody sufficient to bind an antigen. In certain embodiments, the antigen-binding region contains a VHH domain.

[0211] The term “VHH” or “VHH domain” as used herein refers to the antigen- binding portion of a single-domain antibody (sdAb), such as a camelid antibody or shark antibody. In certain embodiments, a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In certain embodiments, a VHH may be truncated at the N-terminus or C-terminus such that it comprises only a partial FR1 and / or FR4, or lacks one or both of FR1 and FR4, so long as the VHH substantially maintains antigen binding and specificity. Safarzadeh et al. Nanobody-based CAR-T cells for cancer immunotherapy. Biomark Res. 10, 24 (2022).

[0212] A single domain antibody (sdAb) refers to an antibody comprising at least one monomeric domain (such as a VHH domain) without a light chain, and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides wherein each polypeptide comprises at least one VHH domain and an Fc region.

[0213] The term “CDR” denotes a complementarity determining region as defined by, e.g., the Kabat numbering scheme, the Chothia numbering schemes, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition.

[0214] A humanized VHH may refer to a VHH in which one or more framework regions have been substantially replaced with human framework regions. In some instances, the humanized VHH can comprise residues that are found neither in the original VHH nor in the human framework sequences, but are included to further refine and optimize VHH-containing polypeptide performance.

[0215] The term “derived from” refers to structural similarity between a first molecule and asecond molecule and does not include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of a cytoplasmic signaling domain that is derived from CD3zeta, the cytoplasmic signaling domain retains sufficient CD3zeta structure such that it has the required function. It does not mean that, to generate the cytoplasmic signaling domain, one must start with a CD3zeta sequence and make desired modifications, to arrive at the cytoplasmic signaling domain.

[0216] The term “about”, when referring to a value, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value. In certain cases, the term “about” encompasses variations of ±10% from the specified value.

[0217] Chimeric

[0218]

[0219] The terms “chimeric antigen receptor,” or alternatively “CAR”, are used interchangeably throughout and refer to a recombinant or engineered polypeptide comprising, e.g., an extracellular antigen- binding region, a transmembrane domain and an intracellular domain. Lee et al.. Clin. Cancer Res. (2012) 18(10):2780; Jensen et al., Immunol Rev. (2014) 257(1): 127. In one embodiment, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule. The costimulatory region may also be 4-1BB (i.e.. CD137), CD27 and / or CD28 or fragments of those molecules. In another aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding region, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. The CAR may comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. Alternatively, the CAR may comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. The CAR may also comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising at least two functionalsignaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. The antigen-binding region of the CAR may contain any antigen-binding antibody fragment. The antibody fragment can comprise one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations of any of the foregoing.

[0220] In certain embodiments, the present CAR is a split CAR system. The split CAR approach is described in U.S. Patent No. 12,383,601, U.S. Patent No. 9,365,641 and U.S. Patent No.

[0221] 10,117,896, incorporated herein by reference. For example, a split CAR system may contain a first CAR having a first antigen-binding region, optionally a hinge region, optionally a transmembrane domain, and a cytoplasmic signaling domain (e.g., CD3 zeta), and a second CAR having a second antigen-binding region, optionally a hinge region, optionally a transmembrane domain, and a costimulatory region (e.g., 4-1BB). When the cell encounters the second antigen, the costimulatory region is activated, and the cell proliferates. When the cell encounters the first antigen, the cytoplasmic signaling domain is activated and cell-killing activity begins. Thus, the CAR-expressing cell is only fully activated in the presence of both antigens. In certain embodiments, the first antigen-binding region recognizes CLL-1, and the second antigen-binding region recognizes CD33. In certain embodiments, the first antigen-binding region recognizes CD33, and the second antigen-binding region recognizes CLL-1.

[0222] In certain embodiments, the domains in the CAR are in the same polypeptide chain. In certain embodiments, the domains in the CAR are not contiguous with each other, for example, are in different polypeptide chains.

[0223] In certain embodiments, the first CAR (component) is encoded by a first polynucleotide, and the second CAR (component) is encoded by a second polynucleotide. In certain embodiments, the first and second polynucleotides are on a single nucleic acid molecule. In certain embodiments, the first and second polynucleotides are on separate nucleic acid molecules. In an embodiment, the polynucleotide is DNA or RNA. In certain embodiments, the first and second polynucleotides are situated in the same orientation, e.g., transcription of the first and second polynucleotides proceeds in the same direction. In certain embodiments, the first and second polynucleotides are situated in different orientations. In certain embodiments, a single promoter controls expression of the first and second polynucleotides.

[0224] In certain embodiments, the first and second polynucleotides are on a single nucleicmolecule in the same frame and translated as a single polypeptide.

[0225] Production of the first CAR (component) and the second CAR (component) can be achieved, e.g., by using separate promoters, or by the use of a bicistronic transcription product (which can result in the production of two proteins by cleavage of a single translation product or by the translation of two separate protein products). In certain embodiments, a first promoter controls expression of the first polynucleotide and a second promoter controls expression of the second polynucleotide.

[0226] A CAR described herein may be encoded by a single nucleic acid and expressed as a single polypeptide, which is subsequently cleaved into different polypeptides, each representing a distinct functional unit. In some embodiments, where the CAR contains two or more functional polypeptide units, the different functional units may be co-expressed using one or more polynucleotide chains. In one embodiment, co- stimulation is provided by an accessory module that is co-expressed with the CAR but is not an integral part of the CAR polypeptide.

[0227] In certain embodiments, the oligonucleotide encoding a linker peptide is placed such that a cell can express a fusion protein comprising the first CAR (component) and the second CAR (component), and the fusion protein is subsequently processed into two peptides. In some embodiments, the first polynucleotide is upstream (at the 5’ end) of the second polynucleotide. In some embodiments, the second polynucleotide is upstream (at the 5’ end) of the first polynucleotide.

[0228] In certain embodiments, an oligonucleotide encoding a linker peptide is situated between the first and second polynucleotides. In one embodiment, the first polynucleotide and the second polynucleotide are separated by an oligonucleotide encoding a linker. In certain embodiments, the first polypeptide and the second polypeptide are expressed as a single polypeptide separated by a linker.

[0229] The linker may be cleavable or non-cleavable.

[0230] Examples of cleavable peptides include, but are not limited to, self-cleaving viral 2A peptides, for example, a porcine teschovirus-1 (P2A) peptide, a Thosea asigna virus (T2A) peptide, an equine rhinitis A virus (E2A) peptide, a foot-and-mouth disease virus (F2A) peptide, or combinations, variants and functional equivalents thereof. Self-cleaving 2A peptides allow expression of multiple gene products from a single construct. See, for example, Chang et al., Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells, MAbs7(2): 403-412 (2015).

[0231] In certain embodiments, the linker comprises a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. In certain embodiments, the linker comprises a P2A peptide.

[0232] Non-cleavable linkers may be a flexible linker composed of flexible residues which allow freedom of motion of adjacent protein domains relative to one another. Non-limiting examples of such residues include glycine and serine. A flexible linker may refer to a peptide linker that contains amino acids such as glycine and / or serine residues used alone or in combination. In one embodiment, the flexible linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1. For example, n=l, n=2, n=3. n=4, n=5 and n=6, n=7, n=8. n=9 and n=10. In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3. In some embodiments, linkers include non-flexible residues.

[0233] In an embodiment, a Ser-Gly-Ser-Gly (SGSG) or GSG motif is also added upstream of the cleavable linker sequences to enhance the efficiency of cleavage. In some embodiments, a furine cleavage site (RAKR) is added upstream of the SGSG or GSG motif to facilitate cleavage of the 2A peptide following translation.

[0234] The first and second polynucleotides (e.g., encoding the different units of a CAR) may be linked by IRES (Internal Ribosomal Entry Site) sequences. Alternately, the first and second polynucleotides (e.g., the different functional units of a CAR) are encoded by two different polynucleotides that are not linked via a linker but are instead encoded by, for example, two different nucleic acids or vectors.

[0235] The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta” may be the protein provided as GenBank accession numbers NP_932170, NP_000725. or XP_011508447: or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, and a “zeta stimulatory domain” or alternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatory domain” may be the amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation.

[0236] A chimeric receptor may refer to a non-naturally occurring molecule that can be expressed on the surface of a host cell and comprises an antigen-binding fragment that binds to an antigen.In addition to the antigen-binding fragment, the chimeric receptor may further comprise one or more of a hinge region, a transmembrane domain, at least one co-stimulatory region, and a cytoplasmic signaling domain. In some embodiments, the chimeric antigen receptor comprises from N terminus to C terminus, an antigen-binding region (or fragment), a hinge region, a transmembrane domain, and a cytoplasmic signaling domain. In some embodiments, the chimeric antigen receptor further comprises at least one co-stimulatory region. Thus, the chimeric antigen receptor may comprise from N terminus to C terminus, an antigen-binding region (or fragment), a hinge region, a transmembrane domain, a co-stimulatory region, and a cytoplasmic signaling domain.

[0237] In some embodiments, the chimeric antigen receptors comprise a hinge region, which may be located between the antigen-binding region and a transmembrane domain. The hinge region may contain about 10-200 amino acids, e.g., 15-150 amino acids, 20-100 amino acids, or 30-60 amino acids. In some embodiments, the hinge region may be of about 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, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids in length. The hinge region may contain 0-300 amino acids, 2 amino acids to 100 amino acids, 5 amino acids to 80 amino acids, 10 amino acids to 60 amino acids, 10 amino acids to 15 amino acids, 20 amino acids to 80 amino acids, 30 amino acids to 70 amino acids, 40 amino acids to 60 amino acids, 50 amino acids to 60 amino acids, or 30 amino acids to 60 amino acids.

[0238] In some embodiments, the hinge region 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 antigen receptors. In some embodiments, the hinge domain is of CD8a or CD28a. In some embodiments, the hinge domain is a portion of the hinge domain of CD8a, e.g., a fragment containing at least 15 (e.g., 20. 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8a or CD28a.

[0239] Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibody, are also compatible for use in the chimeric antigen receptors. In some embodiments, the hinge region is the hinge domain that joins the constant domains CHI and CH2 of an antibody. In some embodiments, the hinge region 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 region comprises the hinge domain of an antibody and the CH3 constant region of the antibody.In some embodiments, the hinge region 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 IgGl, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG4 antibody.

[0240] The hinge region may be a non-naturally occurring peptide. In some embodiments, the hinge region between the extracellular antigen-binding domain and the transmembrane domain is a peptide hinge, such as a (GlyxSer)n (or (GxS)n) hinge, 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.

[0241] Additional peptides that may be used in a hinge region of the chimeric receptors described herein are known in the art. See, e.g., Wriggers et al. Current Trends in Peptide Science (2005) 80(6): 736-746 and PCT Publication WO 2012 / 088461.

[0242] In some embodiments, the chimeric antigen receptors may comprise a transmembrane domain. The transmembrane domain can be in any form known in the art. Transmembrane domains compatible for use in the chimeric antigen receptors may be obtained from a naturally occurring protein. Alternatively, the transmembrane domain may be a synthetic, non-naturally occurring protein segment, e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0243] In some embodiments, the transmembrane domain is that of CD8a. In some embodiments, the transmembrane domain is that of CD28. In some embodiments, the transmembrane domain is that of ICOS.

[0244] In some embodiments, the chimeric antigen receptors comprise one or more costimulatory regions. A co-stimulatory region may be 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 co-stimulatory region of the chimeric antigen receptor can be from a protein which transduces a signal and modulates responses mediated by immune cells, such as T cells, natural killer (NK) cells, macrophages, neutrophils, or eosinophils.

[0245] In some embodiments, the chimeric antigen receptor comprises one or more than one (at least 2, 3, 4, or more) co-stimulatory region. In some embodiments, the chimeric antigenreceptor comprises more than one co-stimulatory region obtained from different proteins. In some embodiments, the chimeric antigen receptor does not comprise a co-stimulatory region.

[0246] Examples of co-stimulatory regions for use in the chimeric antigen receptors can be a domain from co-stimulatory proteins, including, without limitation, CD27, CD28, 4- IBB, 0X40, CD30, Cd40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NK.G2C. B7-H3. In some embodiments, the co-stimulatory region is derived from 4-1BB, CD28, or ICOS. In some embodiments, the co-stimulatory region is derived from CD28 and the chimeric antigen receptor comprises a second co-stimulatory region from 4- IBB or ICOS. In some embodiments, the co-stimulatory region is a fusion domain comprising more than one co-stimulatory region or portions of more than one co-stimulatory region. In some embodiments, the costimulatory region is a fusion of costimulatory regions from CD28 and ICOS.

[0247] In some embodiments, the chimeric antigen receptors comprise a cytoplasmic signaling domain. Any cytoplasmic signaling domain can be used in the chimeric antigen receptors described herein. A cytoplasmic signaling domain may relay a signal, such as interaction of an extracellular ligand-binding domain with its ligand, to stimulate a cellular response, such as inducing an effector function of the cell (e.g., cytotoxicity).

[0248] The chimeric antigen receptors can be prepared by routine methods, such as recombinant technology. Methods for preparing the chimeric antigen receptors may involve generation of a nucleic acid that encodes a polypeptide comprising each of the domains of the chimeric antigen receptors, including the antigen-binding fragment and optionally, the hinge region, the transmembrane domain, at least one co-stimulatory region, and the cytoplasmic signaling domain. In some embodiments, nucleic acids encoding each of the components of the chimeric antigen receptor are joined together using recombinant technology. Sequences of each of the components (e.g., domains) can be joined directly or indirectly (e.g., using a nucleic acid sequence encoding a peptide linker) to form a nucleic acid sequence encoding the chimeric antigen receptor, using methods such as PCR amplification or ligation. Alternatively, the nucleic acid encoding the chimeric antigen receptor may be synthesized. In some embodiments, the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA.

[0249] In one embodiment, the present CAR, from the N-terminus to C-terminus, comprises a signal peptide (also known as leader sequence), an antigen recognition sequence (antigen-bindingdomain), a hinge region, a transmembrane domain, a co-stimulatory region, and a cytoplasmic signaling domain (e.g., a CD3zeta signaling region ( chain portion)).

[0250] As used herein, the terms "CAR-T cell", "CAR-T", "CART", "CART cell” may refer to the T cell that expresses the present CAR.

[0251] The present CAR may be mono-specific. The present CAR may be multi- specific, e.g., bispecific, tri-specific. tetra- specific, etc.

[0252] Immune cells

[0253] The present disclosure also provides immune cells comprising or expressing the first polypeptide and the second polypeptide, immune cells comprising or expressing the recombinant polypeptide, immune cells expressing the present CAR, and immune cells comprising the nucleic acid, the vector, or the system.

[0254] Recognition of a target cell having the antigen(s) on its cell surface by the antigen-binding fragment of the chimeric antigen receptor may transduce an activation signal to the signaling domain(s) (e.g., co-stimulatory region and / or the cytoplasmic signaling domain) of the chimeric antigen receptor, which may activate an effector function in the immune cell expressing the chimeric antigen receptor.

[0255] The chimeric antigen receptor can be introduced into a suitable immune cell for expression via conventional technology. In some embodiments, the immune cells are T cells, such as primary T cells or T cell lines. Alternatively, the immune cells can be natural killer (NK) cells, such as established NK cell lines (e.g., NK-92 cells). In some embodiments, the immune cells are T cells that express CD8 (CD8+) or CD8 and CD4 (CD8+ / CD4+). In some embodiments, the T cells are T cells of an established T cell line, for example, Jurkat cells. Non-limiting examples of immune cells include T cells (e.g., alpha / beta T cells and gamma / delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloic-derived phagocytes.

[0256] Primary T cells may be obtained from any source, such as peripheral blood mononuclear cells (PBMCs), bone marrow, tissues such as spleen, lymph node, thymus, or tumor tissue. In some embodiments, the population of immune cells is derived from a human patient having cancer (e.g., hematopoietic malignancy), such as from the bone marrow or from PBMCs obtained from the patient. In some embodiments, the population of immune cells is derived from a healthy donor. In some embodiments, the immune cells are obtained from the subject to whom the immune cellsexpressing the chimeric antigen receptors will be subsequently administered. Immune cells that are administered to the same subject from which the cells were obtained are referred to as autologous cells, whereas immune cells that are obtained from a subject who is not the subject to whom the cells will be administered may be referred to as allogeneic cells.

[0257] The type of immune cells desired may be expanded within the population of cells obtained by co-incubating the cells with stimulatory molecules, for example, anti-CD3 and anti-CD28 antibodies may be used for expansion of T cells.

[0258] To construct the immune cells that express the chimeric antigen receptors described herein, vectors for stable or transient expression of the chimeric antigen receptor may be constructed via conventional methods as described herein and introduced into immune cells. For example, nucleic acids encoding the chimeric antigen receptors may be cloned into a suitable vector, such as a viral vector.

[0259] In certain embodiments, immune cells (e.g., T cells) are transduced with lentiviral vectors (LVs) encoding the present CAR. The transduced immune cells (e.g., T cells) can target CLL-1 and CD33, synergistically activate the T cells, and induce T cell-mediated immune responses.

[0260] In one embodiment, in the present method, T cells from an autologous patient (or an allogeneic donor) are isolated, activated and genetically modified to generate CAR-T cells expressing the present CAR, and then administered to the patient. CAR-T cells can replicate in vivo resulting in long-term persistence. In addition, the CAR-mediated immune response may be part of an adoptive immunotherapy approach in which the anti-CLL-l / CD33 CAR-T cells elicit an immune response against cells expressing CLL-1 and / or CD33.

[0261] In certain embodiments, cells are isolated from a mammal (e.g.. a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human. The CAR-modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

[0262] The methods of preparing immune cells expressing the present chimeric antigen receptors may comprise activating and / or expanding the immune cells ex vivo. Activating an immune cell means stimulating an immune cell into an activated state in which the cell may be able to perform effector functions (e.g., cytotoxicity). Methods of activating an immune cell will depend on thetype of the immune cell used for expression of the chimeric antigen receptors. Expanding immune cells may involve any method that results in an increase in the number of cells expressing chimeric antigen receptors, for example, allowing the cells to proliferate or stimulating the cells to proliferate. In some embodiments, the cells expressing the chimeric receptors described herein are activated and / or expanded ex vivo prior to administration to a subject.

[0263] The CAR-expressing immune cells may also serve as a vaccine for ex vivo immunization and / or in vivo therapy in a mammal. In addition to using a cell-based vaccine in terms of ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient. Preferably, the mammal is a human. With respect to ex vivo immunization, one or more of the following may occur in vitro prior to administering the cell into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR to the cells, and / or iii) cryopreservation of the cells.

[0264] Antibodies

[0265] The present disclosure provides an antibody comprising the present anti-CLL antigenbinding region, the present anti-CD33 antigen-binding region, the present anti-CLL VHH, the present anti-CD33 VHH, or combinations thereof.

[0266] The present antibody may be mono-specific. The present antibody may be multi-specific, e.g.. bispecific, tri-specific, tetra-specific, etc.

[0267] A bispecific antibody has specificity for one or two antigens. A bispecific antibody molecule is characterized by a first variable domain sequence which has binding specificity for a first epitope and a second variable domain sequence that has binding specificity for a second epitope. In an embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein), such as CLL-1 and CD33. In an embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein), such as CLL-1 or CD33. In an embodiment the first and second epitopes overlap. In an embodiment the first and second epitopes do not overlap. In certain embodiments, a bispecific antibody molecule comprises a first VHH domain which has binding specificity for a first epitope, and a second VHH domain which has binding specificity for a second epitope. In an embodiment, a bispecific antibody molecule comprises a half antibody, or a fragment thereof, having binding specificity for a first epitope, and a half antibody, or a fragmentthereof, having binding specificity for a second epitope.

[0268] Vectors

[0269] The present disclosure provides a nucleic acid encoding the present CAR, the first polypeptide and the second polypeptide, or the recombinant polypeptide. The present disclosure also provides vectors comprising the present nucleic acid.

[0270] The vectors include, but are not limited to, a plasmid, a phagemid, a phage derivative, a virus, and a cosmid.

[0271] The vector may be a viral vector. Viruses, which are useful as vectors, comprise retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In certain embodiments, the present vector is a retroviral vector such as a len ti viral vector. In some embodiments, the vectors for expression of the chimeric antigen receptors are retroviruses. In some embodiments, the vectors for expression of the chimeric antigen receptors are lentiviruses. In some embodiments, the vectors for expression of the chimeric antigen receptors are adeno-associated viruses.

[0272] A variety of promoters can be used for expression of the chimeric receptors, including, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1-alpha (EFl-a) promoter with or without the EFl-a intron. Additional promoters for expression of the chimeric receptors include any constitutively active promoter in an immune cell. Alternatively, any regulatable promoter (e.g., inducible promoters) may be used, such that its expression can be modulated within an immune cell.

[0273] The vector can be introduced into a cell, e.g., mammalian, bacterial, yeast, or insect cell, by any method in the art. For example, the vector can be transferred into a cell by physical, chemical, or biological means.

[0274] Physical methods for introducing a polynucleotide into a cell comprise calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).Biological methods for introducing a polynucleotide of interest into a cell comprise the use of DNA and RNA vectors. Viral vectors can be derived from retroviruses, lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like.

[0275] Chemical methods for introducing a polynucleotide into a host cell comprise colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0276] In some embodiments, the vector (nucleic acid) encoding the chimeric antigen receptor is a DNA vector and may be electroporated to immune cells (see, e.g., Till, et al. Blood (2012) 119(17): 3940-3950). In some embodiments, the vector (nucleic acid) encoding the chimeric antigen receptor is an RNA molecule, which may be electroporated to immune cells.

[0277] Any of the vectors comprising a nucleic acid that encodes a chimeric antigen receptor described herein is also within the scope of the present disclosure. Such a vector may be delivered into host cells such as immune cells by a suitable method. Methods of delivering vectors to immune cells are well known in the art and may include DNA, RNA, or transposon electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA, RNA, or transposons; delivery of DNA, RNA, or transposons or protein by mechanical deformation (see, e.g., Sharei et al. PNAS (2013) 110(6): 2082-2087): or viral transduction. In some embodiments, the vectors for expression of the chimeric receptors are delivered to cells by viral transduction.

[0278] In examples in which the vectors encoding chimeric antigen receptors are introduced to the host cells using a viral vector, viral particles that are capable of infecting the immune cells and carry the vector may be produced by any method known in the art. The viral particles are harvested from the cell culture supernatant and may be isolated and / or purified prior to contacting the viral particles with the immune cells.

[0279] Pharmaceutical compositions

[0280] The present disclosure provides a pharmaceutical composition comprising the present immune cells, the present CAR, the present first and second polypeptides, the present recombinant polypeptide, the present first and second polynucleotides, the present nucleic acid, the present system, or the present vector. The present pharmaceutical composition may further comprise apharmaceutically acceptable carrier, diluent or excipient. Tn one embodiment, the preparation is a liquid preparation, hi one embodiment, the concentration of the immune cells (e.g., CAR-T cells) in the preparation is 1 x 103-lxl08cells / mL, or 1 x 104-lxl07cells / mL.

[0281] Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of a disease or disorder in the subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0282] Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g. Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0283] The present pharmaceutical composition may be delivered to a cell by contacting the cell with the present pharmaceutical composition.

[0284] The present pharmaceutical composition may be delivered / administered to a subject by any route, including, without limitation, intravenous, intracerebroventricular (ICV) injection, intracisternal injection or infusion, oral, transdermal, ocular, intraperitoneal, subcutaneous, implant, sublingual, subcutaneous, intramuscular, rectal, mucosal, ophthalmic, intrathecal, intraarticular, intra-arterial, sub-arachinoid. bronchial and lymphatic administration. The present pharmaceutical composition may be administered parenterally or systemically. The present composition may be administered locally. The pharmaceutical composition may be formulated for intravenous administration.

[0285] The administration of the present compositions may be carried out in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions may be administered to a patient subcutaneously, intradermally,intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (z.v.) injection, or intraperitoneally. In one embodiment, the compositions are administered to a subject (e.g., a patient) by intradermal or subcutaneous injection. In another embodiment, the compositions are administered by i.v. injection. The compositions may be injected directly into a tumor, lymph node, or site of disorder.

[0286] The present immune cells or pharmaceutical composition may be delivered / administered to a subject via intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration, such as by injection or infusion.

[0287] The present pharmaceutical compositions may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.

[0288] When "an effective amount", "a therapeutically effective amount", or "a therapeutic amount" is indicated, the precise amount of the compositions to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). A pharmaceutical composition comprising the immune cells may be administered at a dosage of 104to 109cells / kg body weight, or 105to 106cells / kg body weight, including all integer values within those ranges. The compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0289] The dosage of the above treatments to be administered to a patient may vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for patient administration can be performed according to art-accepted practices. In one embodiment, IxlO6to IxlO10of the immune cells (e.g., CAR-T cells) can be administered to a patient by means of, for example, intravenous infusion for each treatment or each course of treatment.Conditions to be treated

[0290] The present CAR, immune cells or pharmaceutical composition may be used to treat cancer or tumor. The present CAR, immune cells or pharmaceutical composition may be used to treat an inflammatory disease or an immunological disease. The immunological disease may be an autoimmune disorder.

[0291] Also encompassed by the present disclosure is a method of treating cancer. The method may comprise administering the present nucleic acid, system, recombinant polypeptides, CAR, immune cells or pharmaceutical composition to a subject in need thereof.

[0292] The cancer may be a hematologic cancer. The cancer may be a plasma-cell malignancy. The cancer may be plasma cell leukemia. The cancer may be a B-cell malignancy. The B-cell malignancy may be acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), B-cell leukemia, or B cell lymphoma.

[0293] The cancer may be Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, and / or multiple myeloma (MM).

[0294] The cancer may be acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), chronic myeloproliferative disorder, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), myelomonocytic leukemia (M4), acute monocytic leukemia (M5), acute promyelocytic leukemia (M3), or a combination thereof.

[0295] The cancer may be lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell-follicular lymphoma, large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), Marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome. non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma,splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemiavariant, lymphoplasmacytic lymphoma, a heavy chain disease, plasma cell myeloma, solitary plasmocytoma of bone, extraosseous plasmocytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma. ALK+ large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or unclassifiable lymphoma. In some embodiments, the cancer is a relapsed and / or refractory cancer.

[0296] Diseases that may be treated using the present CAR, immune cells or pharmaceutical composition include non-solid tumors (such as hematological cancer, for example, leukemias and lymphomas) or solid tumors. Tumors or cancers to be treated with present CAR, immune cells or pharmaceutical composition include, but are not limited to, carcinoma, blastoma, and sarcoma, and leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, renal cancer, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are included.

[0297] Hematologic cancers are cancers of blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, e.g., acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0298] The cancer may be a solid tumor. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, andlymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer and ovarian cancer.

[0299] The present disclosure provides for a method of treating an inflammatory disease or an immunological disease. The present disclosure provides for a method of treating an autoimmune disease / disorder. The method may comprise administering the present nucleic acid, system, recombinant polypeptides, CAR, immune cells or pharmaceutical composition to a subject in need thereof.

[0300] Inflammatory and / or immunological diseases may involve immune system dysfunction where chronic inflammation causes tissue damage or autoimmune reactions, impacting various organs.

[0301] Non-limiting examples of autoimmune disorders include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE; lupus), myasthenia gravis, inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1 diabetes, Hashimoto’s thyroiditis. Graves’ disease, psoriasis / psoriatic arthritis, celiac disease, Sjogren’s syndrome, vasculitis, alopecia areata, Guillain-Barre syndrome, scleroderma, autoimmune hepatitis, and vitiligo.

[0302] The autoimmune disorder may be systemic lupus erythematosus (SLE) (e.g., lupus nephritis), systemic vasculitis, systemic sclerosis (SSc), inflammatory myopathy (e.g., polymyositis, dermatomyositis, or inclusion-body myositis), systemic scleroderma, multiple sclerosis, myasthenia gravis, a myositis autoantibody-driven disease, or neuromyelitis optica (NMO).

[0303] The autoimmune disorder may be polymyositis, dermatomyositis, or inclusion-body myositis. The autoimmune disorder may be lupus nephritis.

[0304] The autoimmune disorder may be systemic lupus erythematosus (SLE), lupus nephritis (LN), systemic sclerosis (SSc). CREST syndrome (calcinosis, Raynaud’s syndrome, esophageal dysmotility, sclerodactyl, and telangiectasia), opsoclonus, inflammatory myopathy (e.g., polymyositis, dermatomyositis, and inclusion-body myositis), myositis autoantibody-driven diseases, systemic scleroderma, primary biliary cirrhosis, celiac disease (e.g., gluten sensitive enteropathy), dermatitis herpetiformis, Miller-Fisher Syndrome, acute motor axonal neuropathy (AMAN), multifocal motor neuropathy with conduction block, autoimmune hepatitis, antiphospholipid syndrome, Wegener’s granulomatosis, microscopic polyangiitis, Churg-Strausssyndrome, rheumatoid arthritis, chronic autoimmune hepatitis, scleromyositis, myasthenia gravis (MG), Lambert-Eaton myasthenic syndrome, Hashimoto’s thyroiditis, Graves’ disease, Paraneoplastic cerebellar degeneration, Stiff person syndrome, limbic encephalitis. Isaacs Syndrome, Sydenham’s chorea, pediatric autoimmune neuropsychiatric disease associated with Streptococcus (PANDAS), encephalitis, diabetes mellitus type 1, neuromyelitis optica (NMO), chronic inflammatory bowel disease. Hashimoto's disease, organ transplant rejection, and / or neuromyelitis optica spectrum disorder (NMOSD).

[0305] The autoimmune disorder may be pernicious anemia, Addison’s disease, psoriasis, inflammatory bowel disease (IBD), psoriatic arthritis, Sjogren’s syndrome, lupus erythematosus (e.g., discoid lupus erythematosus, drug-induced lupus erythematosus, and neonatal lupus erythematosus), multiple sclerosis, and / or reactive arthritis.

[0306] The autoimmune disorder may be polymyositis, dermatomyositis, multiple endocrine failure, Schmidt’s syndrome, autoimmune uveitis, adrenalitis, thyroiditis, autoimmune thyroid disease, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, presenile dementia, demyelinating diseases, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressier’s syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, adult onset diabetes mellitus (e.g., type II diabetes), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis. Crohn’s disease, sprue, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, juvenile onset rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture’s syndrome, Chagas’ disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, antiphospholipid syndrome, farmer’s lung, erythema multiforme, post cardiotomy syndrome, Cushing’s syndrome, autoimmune chronic active hepatitis, bird-fancier’s lung, allergic disease, allergic encephalomyelitis, toxic epidermal necrolysis, alopecia. Alport’s syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu’s arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampler’s syndrome, eczema, lymphomatoid granulomatosis, Behcet’s disease, Caplan’s syndrome, Kawasaki’s disease, dengue, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis,Shulman’s syndrome, Felty’s syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch’s cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, human immunodeficiency virus infection, echovirus infection, cardiomyopathy, Alzheimer’s disease, parvovirus infection, rubella virus infection, post vaccination syndromes, congenital rubella infection, Hodgkin’s and non-Hodgkin’s lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma, cryoglobulinemia, Waldenstrom’s macroglobulemia, Epstein-Barr virus infection, mumps, Evan’s syndrome, and / or autoimmune gonadal failure.

[0307] The autoimmune diseases also include, e.g., acute disseminated encephalomyelitis, alopecia areata, antiphospholipid syndrome, autoimmune hepatitis, autoimmune myocarditis, autoimmune pancreatitis, autoimmune polyendocrine syndromesautoimmune uveitis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), type I diabetes mellitus (e.g. , juvenile onset diabetes), multiple sclerosis, scleroderma, ankylosing spondylitis, sarcoid, pemphigus vulgaris, pemphigoid, psoriasis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, Behcet's syndrome, Reiter's disease, Berger's disease, dermatomyositis, polymyositis, antineutrophil cytoplasmic antibody-associated vasculitides (e.g., granulomatosis with polyangiitis (also known as Wegener's granulomatosis), microscopic polyangjitis, and Churg-Strauss syndrome), scleroderma, Sjogren's syndrome, anti-glomerular basement membrane disease (including Goodpasture's syndrome), dilated cardiomyopathy, primary biliary cirrhosis, thyroiditis (e.g., Hashimoto's thyroiditis, Graves' disease), transverse myelitis, allergies, arthritis, fibromyalgia, fibromytosis, lupus, vitiligo, and Guillane-Barre syndrome.

[0308] The autoimmune diseases include inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis, rheumatoid arthritis, Type I diabetes, multiple sclerosis, graft vs. host disease following bone marrow transplantation, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondy loarthropathy, systemic lupus erythematosus, insulin dependent diabetes mellitus, thyroiditis, asthma, psoriasis, dermatitis scleroderma, atopic dermatitis, graft versus host disease, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch- Schoenlejn purpurea, microscopicvasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, polyglandular deficiency type I syndrome and polyglandular deficiency type II syndrome, Schmidt's syndrorme, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthopathy, arthropathy, Reiter's disease, psoriatic arthropathy, chlamydia, yersinia and salmonella associated arthropathy spondyloarhopathy, atheromatous disease / arteriosclerosis, atopic allergy, food allergies, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis / Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis C, common varied immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy, fibrotic lung disease, cryptogenic fibrosing alveolitis, postinflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis / polymyositis associated lung disease, Sjogren's disease associated lung disease, ankylosing spondy litis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, idiopathic leucopenia, autoimmune neutropenia, renal disease NOS, glomerulonephritides, microscopic vasulitis of the kidneys, discoid lupus, erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), insulindependent diabetes mellitus, sympathetic ophthalmia, pulmonary hypertension secondaryto connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatio fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Takayasu's disease / arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo, allergic rhinitis (pollen allergies), anaphylaxis, pet allergies, latex allergies, drug allergies, allergic rhinoconjuctivitis, eosinophilic esophagitis, hypereosinophilic syndrome, eosinophilic gastroenteritis cutaneous lupus erythematosus, eosinophilic esophagitis, hypereosinophilic syndrome, and eosinophilic gastroenteritis.

[0309] The autoimmune disorder may be an inflammatory muscle disease. Inflammatory myopathies are a group of diseases that involve chronic muscle inflammation, muscle weakness, and in some cases, muscle pain. The four main types of chronic, or long-term, inflammatory myopathy are: polymyositis, which affects skeletal muscles (the type involved in body movement) on both sides of the body; dermatomyositis, which causes progressive muscle weakness; inclusion body myositis, which is characterized by slow, progressive muscle weakness and muscle shrinking and loss of muscle; and necrotizing autoimmune myopathy, which involves muscle weakness in the upper and lower body.

[0310] In another embodiment, the autoimmune disease is an autoimmune disease caused by overexpression of B cells (such as lupus erythematosus).

[0311] Kits

[0312] Also within the scope of the present disclosure are kits for use of the present CARs, immune cells, nucleic acids, vectors or pharmaceutical compositions. Such kits may include one or more containers comprising the present CARs, immune cells, nucleic acids, vectors or pharmaceutical compositions.

[0313] In some embodiments, the kit can comprise instructions for use in any of the methods described herein. The instructions can comprise a description of administration of the pharmaceutical composition to a subject to achieve the intended activity in a subject. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. In some embodiments, the instructions comprise a description of administering the pharmaceutical compositions to a subject who is in need of thetreatment.

[0314] The instructions relating to the use of the pharmaceutical compositions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.

[0315] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like.

[0316] The following examples of specific aspects for carrying out the present disclosure are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way.

[0317] Example 1

[0318] We adopted a split dual-CAR-T strategy to design our AML CAR-T cell therapy product, which enhances specificity / sensitivity and reduces toxicity by targeting dual tumor- associated antigens (TAAs) (Fesnak et al., Engineered T cells: the promise and challenges of cancer immunotherapy, Nat. Rev. Cancer 16, 566-581 (2016)). Studies have shown thatCLL-1 andCD33 are expressed at high levels in both AML blast cells and LSCs (Willier et al., CLEC12A and CD33 coexpression as a preferential target for pediatric AML combinatorial immunotherapy. Blood 137, 1037-1049 (2021)). We chose CLL-1 as our primary TAA, and CD33 as the secondary TAA.

[0319] The lead CLL-l / CD33-specific dual-CAR contains an anti-CLL-1 CAR (a CLL-1 -specific binding fragment of heavy chain only antibody (VHH) / a CD3(^ signaling domain-containing first-generation CAR) and an anti-CD33 CAR (a CD33-directed VHH binding fragment / a 4- IBB costimulatory domain-containing chimeric co-stimulatory receptor, CCR). This design allows the dual-CAR T cells to show the highest activity only under the presence of both antigens and thus possess improved safety with minimized parallel reactivity against normal tissues bearing a single antigen.

[0320] The dual-CAR T cells can be used for the treatment of relapsed or refractory acute myeloid leukemia (R / R AML). These CLL-l / CD33-directed CAR T cells can be manufactured using a patient’s own T cells and a third- generation self-inactivating lentiviral vector which encodes a split dual-CAR molecule. This dual-targeting approach potentiates the therapeutic efficacy of the lead dual-CAR T cells against CLL-l / CD33-double positive AML blasts and leukemia stem cells whileenhancing safety by reduced toxicity against normal tissues bearing a single antigen.

[0321] To confirm the simultaneous CAR expression in the lead dual-CAR T cells and their differential potency against CLL-1 / CD33 single-positive versus double-positive antigens, antigen binding assay using recombinant antigen proteins and coculture assays with CLL-1 / CD33 doublepositive wild type and single-knockout AML cells were performed.

[0322] Specifically, to evaluate the activity of the lead dual-CAR T-cell product, human AML cell lines (HL-60-Luc2 and U-937-GFP-Luc2, both CLL-1+CD33+) underwent CRISPR / Cas9-mediated gene editing to generate CLL-1 / CD33 single knockout (CLL-1+CD33 or CLL-1'CD33+) and double knockout (CLL-1 CD33 ) single clones. The lead dual-CAR T cells in co-culture experiments released the highest levels of IFNy only upon activation by CLL- 1+CD33+AML cells rather than the single antigen-positive target cells. In addition, luciferase killing assays demonstrated that these dual-CAR T cells exhibited strong cytotoxic activity against CLL-1+CD33+AML cells, while showing weaker cytotoxic activity against CLL-1 / CD33 single-positive AML cells. Therefore, the lead dual-CAR T cells display differential potency against single antigen-expressing targets versus dual antigens-expressing targets. This in vitro study supports the efficacy and safety of dual-CAR T cells with the well-selected CLL-1 CAR and CD33 CCR for clinical trials.

[0323] Results

[0324] Through optimized design, CAR-T cells can show strong activity only when specific dual TAAs are co-expressed (Hanada et al., Double or nothing on cancer immunotherapy, Nat. Biotechnol. 31, 33-34 (2013); Kloss et al., Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells, Nat. Biotechnol. 31, 71-75 (2013)). The dual-CAR T cells express two kinds of CARs, one of which recognizes and binds to one TAA and transmits only the CD3^ primary signal, while the other recognizes another distinct TAA and transmits the CD28 / 4-1BB co-stimulatory signal (also called chimeric co-stimulatory receptor, CCR). Therefore, dual-CAR T cells show activity only under the presence of both targets on the tumor and thus possess improved safety (Figure 1A). The dual-CAR approach potentiates the therapeutic efficacy of CAR-T cells against cancers while minimizing parallel reactivity against normal tissues bearing a single antigen.

[0325] We selected CLL-1 as the primary target to construct the first-generation CAR, and CD33as the secondary target to construct the CCR (Figure IB). The two main parts (CLL-1 CAR and CD33 CCR) are separated by the P2A self-cleaving peptide to achieve the expression of two different proteins from one single open reading frame (Figure 1C) (Liu et al., Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector, Sci. Rep. 7, 2193 (2017)). Our early exploration using benchmark anti-CLL-l / CD33 antibody sequences showed that, unexpectedly, the dual-CAR T cells (such as D3, D4, and DC-3) demonstrated a significant leakage to CD33 single positive targets, as evidenced by high levels of IFNy production upon targeting by CLL-T CD33+AML cells (Figures 2A-2D and Figures 3A-3F). To resolve this problem, multiple strategies, including the modifications of antibody sequence combinations, costimulatory domains, CD3 signaling domains, and hinge / transmembrane (H / TM) domains, were employed to test whether differential potency can be achieved. Our efforts resulted in the recognition that the same H / TM domains-mediated dimerization of CLL-1 CAR and CD33 CCR was likely the primary reason for the leakage to CLL-TCD33+targets, for which CAR dimerization was reported in previous publications (Leddon et al., The CD28 Transmembrane Domain Contains an Essential Dimerization Motif, Front Immunol. 11, 1519 (2020); Hirabayashi et al., Dual Targeting CAR-T Cells with Optimal Costimulation and Metabolic Fitness enhance Antitumor Activity and Prevent Escape in Solid Tumors, Nat. Cancer 2, 904-918 (2021); Muller et al, The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerization With CD28, Front. Immunol. 12, 639818 (2021)). Further investigation found that using two alternative H / TM domains sets for CLL-1 CAR and CD33 CCR (e.g., one from CD8, and one from CD28), can prominently abolish the leakage effects, showing remarkably differential potency between CD33 single targeting and CLL-1 / CD33 dual targeting (such as Figure ID and Figure IE). Subsequently, "-28HTM" and "-28-8" were established as the dual-CAR frameworks to develop our dual-CAR products.

[0326] We generated anti-CLL-1 and anti-CD33 heavy chain-only single-chain antibodies (VHHs). Through alpaca immunization, phage panning and screening, and antibody purification and verification, we obtained multiple candidate VHHs (Figure 15). In addition, the antigen binding ability and potency of VHH-containing mono-specific CAR-T cells (CLL-A01 to CLL-A12; CD33-B01 to CD33-B11) were evaluated and compared to substantiate the suitability of these VHHs in a CAR construct (Figures 6A-6F and Figures 9A-9F). As a result, CLL-A04 / A07 / A09 and CD33-B04 / B06 / B11 were identified as lead anti-CLL-1 and anti-CD33 VHHs,respectively. Further comparative tests of the AxBy dual-CAR structures (A04 / A07 / A09 x B04 / B06 / B11, in "-28HTM" and "-28-8" frameworks) were conducted (Figures 10A-10D, Figures 11A-11D and Figures 12A-12D), and anti-CLL-1 VHHs and anti-CD33 VHHs with good specificity and safety were used to construct our dual-targeting CAR structures (Figure 1C).

[0327] Preclinical in vitro studies showed that these dual-CAR T cells displayed differential potency against single antigen-expressing targets versus dual targets-expressing targets (Figure ID and Figure IE), enabling them to effectively kill CLL-1+CD33+AML blast and LSCs while preserving CD33 single positive HSCs and liver cells. In addition, compared with the prevailing second-generation CLL-1 mono-specific CARs, our dual-CARs with a first-generation anti-CLL-1 CAR can potentially reduce the toxicity to CLL-1 single positive normal myeloid cells. Moreover, the dual-CAR with co-targeting of CLL-1 and CD33 can potentially enhance the killing of CLL-llow / " CD33+AML blasts (such as M0LM13 cells in Figure 14), thus significantly reducing the chance of AML relapse or delaying the occurrence of AML relapse. In summary, our dual-CAR cell therapy can be a bridge to alloHSCT, a supplement to traditional therapies, or a standalone therapy for patients with r / r AML to achieve better therapeutic outcomes due to effective recognition and safe elimination of AML.

[0328] A serial of mono-specific CARs and dual-CARs were constructed using benchmark anti-CLL-1 single-chain variable fragment (scFv) CLL-05 and anti-CD33 scFv CD33.3. As shown in Figure 2A, DI and D2 are the CAR constructs targeting CLL-1 and CD33. respectively. Compared with the regular 2ndgeneration CAR, DI lacks the co-stimulatory domain of 4- IBB and D2 lacks the CD3(^ activation domain. The CLL-l / CD33-specific dual-CARs (D3 and D4) were assembled by linking DI and D2 using the P2A peptide in different orientations (D1-P2A-D2 for D3, and D2-P2A-D1 for D4).

[0329] The self-cleaving peptide P2A allows the expression of two separate proteins from a single polycistronic mRNA vector with high cleavage efficiency. Thus, we confirmed that T cells transduced with D3 and D4 lentiviruses had simultaneous expression of DI mono-CAR and D2-mono-CAR, as determined by incubating T cells with human IgG Fc-tagged CLL-1 recombinant protein and His-tagged CD33 recombinant protein (Figure 2B).

[0330] HL-60 cells and U-937 cells are known to express both CLL-1 and CD33 antigens. To facilitate the evaluation of candidate dual-CAR T cells’ differential potency against CLL-1 / CD33 single-positive versus double-positive antigens, these cell lines with modified overexpression ofluciferase gene (HL-60-Luc2) or luciferase gene and GFP gene (U-937-GFP-Luc2) were subjected to gene knock out via the CRISPR-Cas9 gene editing technology. Flow cytometrical analysis of the resulting single clones showed that both cell lines with the desired four genotypes were verified: WT (CLL-1+CD33+), CLL-1 sKO (CLL-LCD33+), CD33 sKO (CLL-HCD33 ), and dKO (CLL-1 CD33 ). These cell lines displayed a purity percentage of desired CLL-1 / CD33 expression pattern as high as -99% (Figure 2C).

[0331] Subsequent T cells and AML cells co-culturing assays demonstrated that the D3 dual-CAR T cells produced higher levels of cytotoxic cytokine IFNy upon activation by WT (CLL-1+CD33+) target cells than those activated by antigen-single positive target cells (CLL-1+CD33 or CLL-1 CD33+) (Figure 2D). As expected, D2 T cells produced background levels of IFNy upon the encounter of CD33+target cells due to the lack of CD3y activation domain. DI T cells produced large amounts of IFNy when activated by CLL-1+target cells as DI is the 1stgeneration CAR. However, D3 and D4 dual-CAR T cells also released large amounts of IFNy when activated by the CLL-1‘CD33+cells (Figure 2D), suggesting an unexpected leakage to CD33-single positive targets. This leakage seemed to be more severe for the D4 dual-CAR T cells, probably due to more expression of CD33-CAR in D4 CAR-T cells than that in D3 CAR-T cells (Figure 2B). It is worth noting that WT (CLL-1+CD33+) target cells-activated D3 and D4 dual-CAR T cells produced much more IFNy than CD33 sKO (CLL-1+CD33 ) cells-activated D3 and D4 dual-CAR T cells (Figure 2D), implying the additive or synergistic effects of dual- targeting can enhance dual-CAR T cell potency.

[0332] To increase the lentivirus transduction efficiency through reducing the vector size, we decided to use the smaller- sized binder VHH (heavy chain only antibody) to replace the scFvs in dual-CAR constructs. In addition, the orientation of two mono-CARs (D1-P2A-D2) as in D3 dual-CAR was selected for further optimization, as D3 demonstrated less leakage than D4 to CD33-single positive targets (Figure 2D). This resulted in the new DC-3 dual-CAR design using the benchmark anti-CLL- 1 VHH (CLL- 11) and anti-CD33 VHH (CD33.16) (Figure 3A). We noticed that previous CAR design utilized hinge / transmembrane (H / TM) domains from CD8a for both DI and D2, which can cause CAR dimerization. Therefore, we replaced the second H / TM domains of CD8a from DC-3 CAR with the H / TM domains of CD28 to generate a new CAR construct DC-3-28HTM (Figure 3A). The secondary-generation mono-specific CARs targeting CLL-1 (CLL-11) and targeting CD33 (CD33.7) were used as controls to confirm the expression of antigens intargets.

[0333] Antigen binding assay showed that both DC-3 and DC-3-28HTM dual-CAR T cells had simultaneous expression of CLL-1 CAR and CD33 CAR (also named CD33 CCR) (Figure 3B).

[0334] Co-culture cytokine release assays demonstrated that DC-3 had evident leakage to CLL-LCD33+AML cells, while DC-3-28HTM showed drastically reduced IFNy release upon the encounter of CLL-1‘CD33+HL-60 cells (Figure 3C) and U-937 cells (Figure 3D), suggesting that the abrogation of CAR dimerization using different H / TM domains in dual-CAR constructs can substantially eliminate leakage to CD33-single positive targets. Moreover, further luciferase killing assays showed that DC-3-28HTM dual-CAR T cells possessed differential potency in killing CLL-1 / CD33 single positive and double positive HL-60 cells (Figure 3E) and U-937 cells (Figure 3F). This is consistent with the reduced leakage / IFNy production as DC-3-28HTM dual-CAR T cells kills CLL-1+CD33+WT AML cells much more efficiently than the CD33-single positive AML cells.

[0335] Since using alternative hinge / TM domains for CLL-1 CAR and CD33 CCR in a dual-CAR construct led to remarkably reduced leakage to CLL-1 CD33+targets, we tested dual-CAR construct formats with different sets of hinge / TM domains. The "-28HTM" format had H / TM domains from CD8a for CLL-1 CAR before P2A and H / TM domains from CD28 for CD33 CCR after P2A, while the "28-8" format had H / TM domains from CD28 for CLL-1 CAR before P2A and H / TM domains from CD8a for CD33 CCR after P2A (Figure 4A). In addition, to test the impact of signal peptide (SP) on CAR expression and CAR-T potency, we replaced the second CD8 SP of DC-3-28HTM dual-CAR with the SP of CD28 and generated DC-3-8-28 dual-CAR (Figure 4A). All three dual-CAR constructs can render simultaneous CLL-1 CAR and CD33 CCR expression in T cells, as evidenced by antigen binding assays (Figure 4B). Potency evaluation using co-culture cytokine release measurement confirmed that all three dual-CARs were able to significantly reduce leakage to CLL-1 CD33+targets, and the DC-3-8-28 dual-CAR T cells showed slightly decreased levels of IFNy production upon activation by WT targets (Figure 4C and Figure 4D). Therefore, we considered the "-28-8" format as another favorable dual-CAR framework, and this was also supported by DC-3-28-8 dual-CAR T cells’ differential killing of CLL-1 CD33+AML cells and CLL-1+CD33+AML cells in luciferase killing assays (Figure 4E and Figure 4F). We noticed that DC-3-28HTM (Figures 3E, 3F) and DC-3-28-8 (Figure 4E, 4F) dual-CAR T cells appeared to kill CLL-UCD33 AML cells and CLL-1+CD33+AML cellscomparably, although more IFNy was released upon T-cell engagement with the CLL-1+CD33+WT targets. This may be due to that the CLL-1 CD33+AML cells could be more sensitive to T-cell mediated killing signal than the WT AML cells.

[0336] Next, candidate anti-CLL-1 VHHs were obtained through camelid immunization and library screening (Figure 15). A total of 12 unique anti-CLL-1 VHHs were identified and the fusion proteins consisting of the individual VHH and the Fc portion of human IgGl with C220A mutation were produced (Figure 5A). In vitro antigen binding assay using recombinant hCLL-1-His protein was performed, and most anti-CLL-1 VHHs showed an EC50 as low as 0.098 nM to 0.32 nM, except CLL-A11 showed the highest EC50 as 0.834 nM (Figure 5B). Moreover, Surface Plasmon Resonance (SPR) assay was conducted to determine the binding affinity of these VHHs, and they demonstrated KD values from 0.1 nM to 99.8 nM (Figure 5C). Further characterization by incubating candidate VHHs with CLL-1 -overexpressing A549 cells (Figure 5D), HL-60 cells (Figure 5E) and U-937 cells (Figure 5F) showed that these VHHs can bind to tested cell lines.

[0337] Further characterization of anti-CLL-1 VHHs were performed through using these VHHs as binders in a 2ndgeneration mono-CAR format with SP / H / TM domains from CD8a, 4- IBB costimulatory domain and CD3(^ signaling domain (Figure 6A). Antigen binding assays with CLL-1-hFc recombinant protein demonstrated that the CLL-Ax mono-CARs had good CAR expression as the positive control CARs with a benchmark binder (CLL-06, CLL-10, and CLL-11) (Figure 6B). These CLL-Ax CAR-T cells were co-cultured with WT HL-60 cells (Figure 6C). CLL-1 single knock out (sKO) HL-60 cells (Figure 6D), WT U-937 cells (Figure 6E), and CLL-1 sKO U-937 cells (Figure 6F). IFNy quantitation results demonstrated that most candidate VHHs released a large amount of IFNy upon CLL-1 antigen engagement, and CLL-A02, CLL-A03 showed lower levels of IFNy production. In addition, except CLL-A12, which produced IFNy at -700 pg / mL upon encounter of CLL-l-sKO U-937 cells, other CLL-Ax (CLL-A01 to CLL-A11) CARs showed good specificity (Figure 6F). Subsequent luciferase killing assays confirmed the superior potency of all candidate CLL-Ax CAR-T cells in killing HL-60 cells (Figure 7A) and U-937 cells (Figure 7B).

[0338] Similar characterizations of anti-CD33 VHHs were performed using CD33-Bx recombinant fusion protein (Figures 8A-8F) and CD33-Bx CAR-T cells (Figures 9A-9F). The CD33-Bx recombinant proteins consisted of the individual VHH and the Fc portion of human IgGl with C220A mutation (Figure 8A). The EC50 values of 11 anti-CD33 VHHs, including CD33-B01to CD33-B11, were determined by ELISA, and these VHHs showed an EC50 as low as 0.096 nM to 0.660 nM, except CD33-B08 showed the highest EC50 as 1.639 nM (Figure 8B). SPR assay determined that these VHHs had good binding affinity with KD values from 0.4 nM to 92.5 nM (Figure 8C). Additional characterization by incubating candidate VHHs with CD33-0 verexpressing A549 cells (Figure 8D), HL-60 cells (Figure 8E) and U-937 cells (Figure 8F) showed that most VHHs can bind well to tested cell lines, except that CD33-B07 and CD33-B08 had poor binding to CD33-OE A549 cells.

[0339] The applicability of CD33-Bx VHHs as a binder in a CAR construct was also evaluated in the same 2udgeneration mono-CAR format (Figure 9A). As the positive control CARs with a benchmark binder (CD33.7, CD33.16, and CD33.17), all the CD33-Bx CAR-T cells showed good antigen binding ability (Figure 9B). These CD33-Bx CAR-T cells were co-cultured with WT HL-60 cells (Figure 9C), CD33 sKO HL-60 cells (Figure 9D), WT U-937 cells (Figure 9E), and CD33 sKO U-937 cells (Figure 9F). IFNy quantitation results demonstrated that most candidate VHHs released a large amount of IFNy upon CD33 antigen engagement, and CD33-B02, CD33-B03, CD33-B10, and CD33-B11 showed lower levels of IFNy production. In addition, except CD33-B07 and CD33-B08, which produced IFNy at -500 pg / mL and -1400 pg / mL upon encounter of CD33 sKO HL-60 cells and CD33 sKO U-937 cells, respectively, other CD33-Bx CARs (CD33-B01 to CD33-B06, CD33-B09 to CD33-B11) showed good specificity (Figure 9F).

[0340] Through comparing the antigen binding ability and potency of VHH-containing mono-specific CAR-T cells (CLL-A01 to CLL-A12; CD33-B01 to CD33-B11), we substantiated the suitability of these VHHs in a dual-CAR construct. As a result, CLL-A04 / A07 / A09 and CD33-B04 / B06 / B11 were identified as lead anti-CLL-1 and anti-CD33 VHHs, respectively. We next performed the comparative tests of the AxBy dual-CAR structures (A04 / A07 / A09 x B04 / B06 / B 11) in the "-28HTM" framework, in which the CLL-1 CAR before P2A consisted of SP of CD8a, CLL-Ax binder, H / TM domains of CD8a and CD3z signaling domain, while the CD33 CCR after P2A included SP of CD8a, CD33-By binder, H / TM domains of CD28 and 4- IBB co-stimulatory domain (Figure 10A). Subsequent antigen binding assays and potency evaluation also included the 2udgeneration mono-CARs with a lead VHH binder (CLL-A04 / A07 / A09 and CD33-B04 / B06 / B11). All the AxBy-28HTM dual-CAR T cells (x=04 / 07 / 09, y=04 / 06 / ll) displayed simultaneous CLL-1 CAR and CD33-CCR expression, as evidenced by binding of CLL-1 -hFc and CD33-His proteins (Figure 10B). Remarkably, all the AxBy-28HTM dual-CAR T cells showeddifferential IFNy release when co-cultured with HL-60 cells (Figure 10C) and U-937 cells (Figure 10D) of four genotypes with minimal leakage to CLL-1 CD33+targets. Notably, our AxBy-28HTM candidates exhibited much higher levels of IFNy release than the benchmark DC-3-28HTM upon activation by WT HL-60 targets, but they still produced very low levels of IFNy after engagement with the CLL-LCD33+HL-60 cells.

[0341] Since we previously identified the "-28-8" framework as another suitable format for dualCAR to achieve differential potency against single antigen- and double antigens-expressing target cells (Figures 4A-4F), we designed new constructs to perform side-by-side comparison of the "-28HTM" and "28-8" frameworks. Additional two dual-CAR constructs (A09B06-28-8 and A09B11-28-8) with CLL-A09 and CD33-B06 or CD33-B11 VHH combinations were compared with the A09B06-28HTM and A09B11-28HTM dual-CARs (Figure 11A). While the "A09By-28HTM" dual-CAR T cells (y=06 or 11) displayed good binding to both CLL-l-hFc protein and CD33-His protein, the "A09By-28-8" dual-CAR T cells exhibited good binding to CD33-His protein but poor binding to CLL-l-hFc protein (Figure 11B). Although these A09By-28-8 T cells showed differential potency in terms of IFNy release upon co-culture with HL-60 cells (Figure 11C) and U-937 cells (Figure 11D) of four genotypes, they produced much lower levels of IFNy than their counterpart "-28HTM" T cells after activation by WT AML cells, most likely due to their poor binding to CLL-1 antigen (Figure 11B). However, our previous results demonstrated that DC-3-28HTM and DC-3-28-8 dual-CAR T cells exhibited comparable antigen binding ability and overall potency (Figures 4A-4F). This divergence implies that different combinations of anti-CLL-1 VHH and anti-CD33 VHH could confer dual-CAR T cells varied ability in recognizing dual antigens simultaneously, which is possibly related to steric hindrance effects caused by overexpressed CAR / CCR and varied epitope accessibility of paired VHHs.

[0342] We chose A07B11-28HTM as one of our lead CLL-l / CD33-specific dual-CAR constructs (Figure 12A) and demonstrated that A07B11-28HTM dual-CAR T cells possess differential potency against CLL-1 / CD33 single-positive AML cells versus double-positive AML cells. First, we confirmed the simultaneous expression of anti-CLL-1 CAR and anti-CD33 CAR in A07B11-28HTM dual-CAR T cells, as measured by the binding of hFc-tagged CLL- 1 recombinant protein and His-tagged CD33 recombinant protein (Figure 12B). The mono-specific 2ndgeneration CARs with the same binder sequence as A07B11-28HTM, directed by CLL-1 (CLL-sCAR) or CD33 (CD33-sCAR), were constructed as controls.IFNy release after coculturing with target cells was used to evaluate the potency of CAR-T cells. The control T (NTD) and three CAR-T (CLL-A07 mono-CAR / CD33-Bll mono-CAR / A07B11-28HTM dual-CAR) groups exhibited drastically different patterns of IFNy production upon activation by disparately expressed CLL-1 / CD33 antigens from HL-60-Luc2 cells and U-937-GFP-Luc2 cells (Figure 12C) of four different genotypes. As expected, the NTD group only produced background levels of IFNy. While CLL- 1- directed and CD33-directed mono-specific CAR-T cells (CLL-A07 and CD33-B11) showed significantly high levels of IFNy release only upon activation by corresponding antigen-positive target cells. A07B11-28HTM group exhibited differential IFNy production in responding to single-positive antigen versus doublepositive antigens, and produced the highest levels of IFNy upon dual-targeting (Figure 12C).

[0343] It is worth noting that mono-CAR T cells (CLL-A07 and CD33-B11) released comparable levels of IFNy between the activation by single-positive antigen (CLL-1+CD33‘ or CLL-FCD33+) and double-positive antigens (CLL-1+CD33+), suggesting that the single KO target cells and wild type target cells had comparable abundance of CLL-1 / CD33 antigen. This excluded the possibility of uneven antigen abundance as the cause of differential IFNy production observed in A07B11-28HTM T cells.

[0344] To verify the differential cytotoxic effect of A07B11-28HTM, the luciferase-based killing assays using AML cells of four genotypes were conducted. After incubating A07B11-28HTM dual-CAR T cells with HL-60-Luc2 cells or U-937-GFP-Luc2 cells (Figure 12D) overnight at different effector-to-target (E: T) ratios, the remaining viable cells were lysed, and the released luciferase was quantified by enzyme activity measurement (luciferase activity is proportional to the number of live target cells). In each cell line, the relative light unit (RLU) value of the group with an E: T ratio of 0:1 (i.e., only AML target cells) was set to 100%. Notably, A07B11-28HTM T cells exhibited strong cytotoxic activity against CLL-1+CD33+wild type and CLL-1 singlepositive AML cells, while showing no or only weak cytotoxic activity against CD33 singlepositive AML cells, even at high E:T ratios (Figure 12D). Therefore, these results indicate A07B11-28HTM dual-CAR T cells can effectively eliminate CLL-1 -positive malignant target cells in vitro and largely avoid the killing of normal cells that only express the CD33 antigen (such as hematopoietic stem cells and hepatocytes).

[0345] To test whether combining a CAR and a CCR can enhance T cell sensitivity to low antigen density, we studied whether the CLL-1 / CD33 dual-CAR T cells with the "-28HTM" and "-28-8"framework were able to kill the tumor cells with a low expression level of the primary antigen CLL-1. M0LM13 is a human AML cell line and has a high level of CD33 expression but a low level of CLL-1 expression (CLL-llow / CD33+). As shown in Figure 14, the 2ndgeneration anti-CD33 mono-CAR T cells (CD33.16) efficiently (100%) eradicated CLL-llow / ’CD33+MOLM13 cells at a higher E:T ratio (1:1), while the 2ndgeneration anti-CLL-1 mono-CAR-T cells (CLL-11) can only eliminate -50% cells. However, the CLL-1 / CD33 dual-targeting dual-CAR T cells (DC-3-28HTM and DC-3-28-8) displayed more efficient (-80%) killing of MOLM13 cells than the CLL-11 CAR-T cells (Figure 14). Therefore, co-targeting of CLL-1 and CD33 can enhance the killing of CLL- llow / CD33+AML blasts, thus potentially significantly reducing the chance of AML relapse or delaying the occurrence of AML relapse. Multiplexing targeting and co-stimulation through the combination of a CLL- 1 -directed CAR and a CD33-directed CCR might be a powerful strategy to enhancing cytotoxic efficacy and improve the clinical outcomes through preventing relapses of AML clones with a low CLL-1 antigen density.

[0346] Conclusions

[0347] The in vitro evaluation by coculturing AxBy-28HTM dual-CAR T cells and human AML cells (HL-60-Luc2 and U-937-GFP-Luc2) with disparately expressed CLL-1 / CD33 antigens showed that A07B11-28HTM T cells exhibited differential potency against single-positive target versus double-positive targets. A07B11-28HTM T cells can effectively eliminate CLL-1 / CD33-double positive target cells while largely sparing the CD33-single positive targets, thus reducing the hematotoxicity and hepatotoxicity caused by CD33 single targeting. This study provides evidence on the superior efficacy and improved safety of CLL-l / CD33-directed dual-CAR T cells.

[0348] Example 2

[0349] of Recombination-deletion

[0350] Recombination-deletion of homologous nucleic acid cassettes in chimeric antigen receptor (CAR) vectors occurs when duplicated DNA sequences (e.g., in multi-cistronic designs) undergo intra- vector deletion, resulting in the loss of one or more transgene components. This phenomenon frequently occurs in retroviral and len ti viral vectors, where high homology between cassettes leads to efficient, unintended deletion, impairing the production of complex "designer T cells" [Im et al., Recombination-Deletion between Homologous Cassettes in Retrovirus Is Suppressed Via aStrategy of Degenerate Codon Substitution." Mol. Ther. Methods Clin. Dev., (2014) 1: 14022. Lam et al., Development of a Bicistronic Anti-CD19 / CD20 Car Construct Including Abrogation of Unexpected Nucleic Acid Sequence Deletions. Mol. Ther. Oncolytics, (2023) 30: 132-49.]

[0351] Key Aspects of Recombination-Deletion in CARs

[0352] Mechanism: When two or more functional domains are highly similar, they can pair during reverse transcription, leading to homologous recombination that deletes the intervening DNA.

[0353] Impact: This reduces the efficacy of, for example, dual-chain CAR T cell therapies by preventing the expression of both designed receptors, as one is often excised.

[0354] Solution: A degenerate codon substitution strategy can be used to minimize sequence homology between repeated domains.

[0355] Effectiveness: Reducing domain homology can significantly suppress the deletion rate to levels indistinguishable from controls, ensuring stable, simultaneous expression of multiple genes.

[0356] This method allows for the engineering of more complex T cell functionalities without the instability caused by sequence repetition, facilitating the development of advanced CAR-based immunotherapies.

[0357] Accumulating studies found that, during the reverse transcription of retroviral and lentiviral vector RNA into transgene DNA of transduced T cells, homologous nucleic acid cassettes in CAR vectors often cause the loss of bicistronic construct transgene DNA. Our previous work identified A07B11-28HTM as one of the leading dual-CAR candidates for treating AML (Figures 12A-12D).

[0358] To decrease the possible loss of A07B11-28HTM construct nucleic acid sequence by recombination-deletion, we optimized the DNA sequence to reduce repeated regions of highly similar sequences within the CAR construct and the whole viral vector.

[0359] The codon-optimized version of A07B11-28HTM (V3.2) involves DNA synonymous substitution in multiple CAR construct domains (CD8a SP, CD8a H / TM, and VHH binder) and shares the same amino acid sequence with the proto A07B11-28HTM.

[0360] Subsequent comparative studies show that V3.2 demonstrated similar patterns of CLL-1 CAR and CD33 CCR expressions as A07B11-28HTM, implying the frequency of transgenes expressed after recombination-deletion in A07B11-28HTM dual-CAR T cells is very low.

[0361] In addition, further functional characterization using two target systems (AML cells withphysiological levels of CLL-1 / CD33 antigen expression and A549 cells with antigen ectopic overexpression) suggests that A07B11-28HTM-V3.2 and A07B11-28HTM exhibit very comparable patterns of activity against the targets of four genotypes (CLL-1 CD33 , CLL-1 CD33+, CLL-1+CD33 , and CLL-1+CD33+).

[0362] Results

[0363] To evaluate whether the codon optimization can affect the antigen binding ability and potency of A07B11-28HTM dual-CAR T cells, we performed side-by-side experiments comparing the A07B11-28HTM and A07B11-28HTM-V3.2 (V3.2) constructs. As shown in Figure 13A, V3.2 demonstrated comparable antigen binding ability to CLL-l-hFc protein and CD33-His protein as A07B11-28HTM, which implies that the frequency of transgenes expressed after potential recombination-deletion in A07B11-28HTM dual-CAR T cells is low.

[0364] In addition, further functional characterizations using two target systems (AML cells with physiological levels of CLL-1 / CD33 antigen expression and A549 cells with antigen ectopic overexpression) were performed. A07B11-28HTM and V3.2 displayed similar differential IFNy release after co-culturing with HL-60 cells and U-937 cells of four genotypes (CLL-1 CD33-, CLL-1 CD33+, CLL-1+CD33 , and CLL-1+CD33+) (Figure 13B), and limited killing of CD33-single positive HL-60 cells (Figure 13C). In addition, these dual-CAR T cells exhibited very limited potency to CD33-OE A549 cells and strong potency to CLL-1 -positive A549 cells (CLL-OE and CLL-1 / CD33 double OE), as evidenced by the co-culture IFNy release assay and the xCELLigence real-time cell analysis (RTCA) assay (Figure 13D).

[0365] Conclusions

[0366] Collectively, A07B11-28HTM-V3.2 and A07B11-28HTM exhibit very comparable patterns of activity against the CLL-l / CD33-expressing targets. The codon-optimized dual-CAR constructs with reduction of recombination-deletion and verified functionality are more favorable for clinical trials.

[0367] Materials and Methods

[0368] Materials

[0369] AML target cells and T cellsHuman AML cell lines HL-60-Luc2 (catalogue number CCL-240-LUC2™) and U-937 (catalogue number CRL- 1593.2™) were originally purchased from the American Type Culture Collection (ATCC). HL-60-Luc2 cells were cultured in Iscove's Modified Dulbecco’s Medium (IMDM) supplemented with 20% (v / v) fetal bovine serum (FBS), 1% (v / v) Penicillin-Streptomycin (100 units / mL penicillin and 100 pg / mL streptomycin), and Blasticidin to a final concentration of 8pg / mL. U-937 cells were cultured in Roswell Park Memorial Institute medium (RPML1640) supplemented with 10% FBS and 1% Penicillin-Streptomycin. All cells were cultured at 37°C and 5% CO2. To create luciferase-expressing target cells, wild type (WT) U-937 cells were infected with GFP-Luc2 overexpression lentivirus, and single clones of U-937-GFP-Luc2 cells were obtained by the limiting dilution method.

[0370] To generate CLL-1 / CD33 single knockout (KO) and double KO AML targets. HL-60-Luc2 cells and U-937-GFP-Luc2 cells (single clone SC-06) were electroporated to deliver the ribonucleoprotein complexes (crRNA: tracrRNA: Cas9) using the Alt-R™ CRISPR-Cas9 System from Integrated DNA Technologies (IDT). CLL-1 crRNA #1 (Hs.Cas9.CLEC12A.l.AA) and CD33 crRNA #3 (CD33_CrRNA_3) were identified to be verified sgRNA / crRNA for knocking out CLL-1 and CD33, respectively. At 5 days after nucleofection, limiting dilution was performed to obtain single clones of HL-60-Luc2 cells and U-937-GFP-Luc2 cells with desired genotypes: WT (CLL-UCD33+), CLL-1 sKO (CLL-1 CD33+), CD33 sKO (CLL-UCD33 ), and dKO (CLL-1 CD33).

[0371] Human T cells were isolated from peripheral blood mononuclear cells (PBMCs) of heathy donors, and the PBMCs were sourced from cryopreserved Leukopaks of HemaCare® (Charles River, catalogue number PB001LCLP4-Kit). T cells were activated with Dynabeads™ Human T-Activator CD3 / CD28 for 3 days and then transduced with CLL-1 CAR / CD33 CCR-expressing lentivirus (AxBy duaLCAR) or left non-transduced (NTD).

[0372] Reagents

[0373] Reagent Name Brand

[0374] BD CBA Human Thl / Th2 Cytokine Kit II BD Biosciences PE anti-human CLL- 1 antibody BioLegend APC anti-human CD33 antibody BioLegend

[0375]

[0376] PE anti-His tag antibody BioLegendAlexa Fluor® 647 AffiniPure™ F(ab')2 Fragment Goat Anti-Human Jackson IgG, Fey fragment specific ImmunoResearch Recombinant Human CLL-1 / CLEC12A Protein (hFc Tag) Sino Biological Recombinant Human Siglec-3 / CD33 Protein (ECD, His Tag), HPLC- Sino Biological verified

[0377] LIVE / DEAD™ Aqua ThermoFisher Recombinant Human IL-2 PeproTech Dynabeads™ Human T Activator CD3 / CD28 ThermoFisher STEMCELL

[0378] EasySep ™ Human T Cell Isolation Kit

[0379] Technologies TrypLE™ Express Enzyme (lx) Gibco

[0380]

[0381] The helper plasmids (CBMG-PRP2-K, CBMG-PRP3-K, and CBMG-PRM2-K) were prepared using Qiagen EndoFree Plasmid Mega Kit (Catalog No. 12981). The purified plasmid samples were analyzed by automated DNA sequencing using Applied Biosystems™ 3730x1 DNA Analyzer and the sequences were verified. The lentiviral vector plasmids AxBy-28HTM dualCAR, AxBy-28-8 dual-CAR, CLL-Ax mono-CAR, and CD33-Bx mono-CAR were prepared, and the sequences were verified by DNA sequencing. Lenti-X™ 293T cell line was purchased from Takara Bio USA, Inc. (Catalog No. 632180).

[0382] Original Plasmid Name CDS

[0383] CBMG-PRP2-K Gag-Pol

[0384] CBMG-PRP3-K Rev

[0385] CBMG-PRM2-K VSV-G

[0386] AxBy-28HTM; AxBy-28-8 Anti-CLL-1 / CD33 dual-CAR

[0387] CLL-Ax Anti-CLL-1 mono-CAR

[0388]

[0389] CD33-Bx Anti-CD33 mono-CAR

[0390] Methods

[0391] Lentivirus preparation

[0392] 293T cells were seeded onto the Poly-D-Lysine pre-treated 6-well plate at a concentration of 2xl0e6 cells in 2 mL complete culture medium (DMEM supplemented with 10% FBS, 100 units / mL penicillin and 100 pg / mL streptomycin) per well. The next day, 293T cells reached a 70%-90% confluency. The vims packaging plasmids, including the shared helper plasmids (CBMG-PRP2-K: 0.78 pg; CBMG-PRP3-K: 0.36 pg; CBMG-PRM2-K: 0.46 pg; per well) andthe individual lentiviral plasmid (dual-CAR or mono-CAR: 2.14 pg; per well), were mixed and transfected into 293T cells using Lipofectamine™ 3000 Transfection Reagent following the manufacturer's protocol. Around 24 hours after plasmids transfection, the old culture medium was replaced with 3 mL complete DMEM culture medium. Around 70 hours after plasmids transfection, culture medium was collected and subjected to spinning down at 1000 g for 10 min at 4°C. The supernatant containing lentiviral particles were further concentrated using Lenti-X™ Concentrator following the manufacturer's protocol, and the aliquots of 50 pL concentrated viral particles were stored at -80°C.

[0393] CAR-T cell preparation

[0394] Human T cells were isolated from the Leukopak with the CliniMACS Prodigy (Miltenyi Biotec) and cryopreserved using CryoStor® CS10. On Day 0, the cryopreserved T cells were thawed, and seeded in a 24- well plate at the concentration of lxl0e6 -2x10e6 cells / mL (2x10e6 - 4xl0e6 cells / well; <2 mL / well) in AIM V medium supplemented with 10% FBS, 100 units / mL penicillin, 100 pg / mL streptomycin, and 200 ZU / mL human IL-2 (complete T cell medium). T cells were activated with Human T-Activator CD3 / CD28 Dynabeads™ (T cell: Bead = 1:1) for 3 days. On Day 3, T cells were infected with lentiviral particles encoding the desired CAR (e.g., dual-CAR and mono-CAR) through spinoculation at 2000 g for 2 hours at 32°C, or left un-infected (non-transduced T cells, NTD). On Day 4 - Day 9, T cells were expanded in complete T cell medium with addition of new medium every day.

[0395] Flow cytometry

[0396] HL-60-Luc2 cells, U-937-GFP-Luc2 cells, and M0LM13 cells (~lxl0e5 cells / test) were stained with PE-conjugated anti-human CLL-1 antibody (2 pL / test) and APC-conjugated antihuman CD33 antibody (2 pL / test) in staining buffer (DPBS supplemented with 2% FBS, 100 pL / test) at 4°C for 25 min.

[0397] Control non-transduced (NTD) T cells and CAR T cells (dual-CAR and mono-CAR, -IxlO5cells / test on Day 5 - Day 7) were washed with DPBS twice (1 mL / tube) to remove serum in the culture medium and then stained with LIVE / DEAD™ Aqua (1:1000 dilution in DPBS) at 4°C for 20 min following the manufacturer's protocol. After washing with DPBS, T cells were incubated with hFc-tagged human CLL-1 recombinant protein (0.1 pg / pL, 1 pL / test) and His-tagged human CD33 recombinant protein (0.1 pg / pL, 1 pL / test) at 4°C for 30 min. Cells were washed with 1 mL staining buffer twice, and further incubated with the following reagents: Alexa Fluor® 647-conjugated goat anti-human IgG AffiniPure™ F(ab')2 fragment (1:100 dilution) and PE anti-His tag antibody (2 pL / test). The volume for the incubation of T cells with the mixed reagents was -100 pL / test. After incubation at 4°C for 25 min, cells were washed with 1 mL staining buffer and subjected to flow analysis. Samples were acquired on the CytoFLEX LX Flow Cytometer. All data were analyzed with the FlowJo V10 software (BD Biosciences).

[0398] For determining the binding ability of anti-CLL-1 or anti-CD33 VHHs to antigenexpressing A549 cells and AML cells (HL-60 and U-937), these target cells were incubated with VHH proteins at the specified concentrations at 4°C for 30 min. After washing with staining buffer twice, cells were further incubated with FITC-conjugated anti-hFc antibody and were subjected to flow cytometrical analysis after washing.

[0399] T cells and target cells co-culturing assays

[0400] Control non-transduced (NTD) T cells and CAR-T cells on Day 6 - Day 9, e.g., Day 8 (4-7 days, e.g., 5 days, after lentivirus transduction) were counted and washed twice with basal AIM V medium (supplemented with 10% FBS, 100 units / mL penicillin, 100 pg / mL streptomycin, and no IL-2) to remove residual IL-2 in the culture medium.

[0401] The pellets of T cells and target cells (HL-60-Luc2, U-937-GFP-Luc2 and A549 cells, each cell line with 4 genotypes: CLL-1+CD33+, CLL-1 CD33+, CLL-1+CD33 , and CLL-1 CD33 ) were resuspended in basal AIM V medium to reach the concentration of 5xl0e5 cells / mL (AML cells) or 2xl0e5 cells / mL (A549 cells).

[0402] AML cells / A549 cells were seeded into a 96-well round / flat-bottom plate at 100 pL / well (5xl0e4 cells / well or 2xl0e4 cells / well) with three replicated wells for each group. T cells (NTD, CLL-1 / CD33 mono-CAR or dual-CAR T) were seeded into the same plate at 100 pL / well (5xl0e4 total or CAR+T cells / well) in 100 pL AIM V basal medium. The wells of the T cell-only group contained 100 pL T cells and 100 pL AIM V basal medium.

[0403] After mixing cells with a multi-channel pipette, T cells and AML cells were co-cultured at 37°C in an incubator for about 22 hours. Cell culture supernatant was collected and frozen as needed at -20°C for subsequent cytokine measurements.Quantification of IFN y in coculture supernatants

[0404] The concentration of cytotoxic cytokine IFNy in co-culture supernatants was measured using BD™ Cytometric Bead Array (CBA) Human Thl / Th2 Cytokine Kit II following the manufacturer's instructions with minor modifications. Briefly, fresh Human Thl / Th2 Cytokine Standards were prepared by diluting the lyophilized proteins with 2 mL Assay Diluent; 100 pL of the Top Standard was further diluted with 100 LIL Assay Diluent to get the 1:2 diluted standard. Then, 1:2 serial dilutions were performed, and a series of standards (top, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, Assay Diluent only) were obtained.

[0405] The supernatants from the co-cultures of CAR-T cells (dual-CAR and CLL-1 mono-CAR) and target cells (e.g., CLL-1+AML cells) were diluted 2-3 folds as needed with the basal AIM V medium, and all other sample groups were undiluted. Samples / Standards, mixed CBA beads, and PE-detection reagents were added in the wells of a 96-well V-bottom plate at the ratio of 1:1:1 (5 pL: 5 pL: 5 pL), and the plate was incubated at room temperature for 3 hours on a rocking shaker. Beads were then washed with 200 pL washing buffer (centrifugation at 200 g, 5 min) and resuspended in 120 pL DPBS. Samples were applied to the CytoFLEX LX Flow Cytometer in Plate Mode, and at least 1000 beads events in the gated beads population were collected for each sample. Flow data were analyzed with FlowJo V10 software, and the mean fluorescence intensity (MFI) values of PE from the Standards were used for plotting the standard curve. The IFNy level of samples was calculated based on the standard curve, and data were plotted using GraphPad Prism 9 (GraphPad Software). Results are shown as the mean ± standard deviation (SD) of three replicates from one representative donor.

[0406] Luciferase killing assay

[0407] The luciferase-expressing AML cells (lOOpL / well, 5xl0e5 cells / well; HL-60-Luc2 and U-937-GFP-luc2, each cell line with 4 genotypes: CLL-1+CD33+. CLL-TCD33+, CLL-PCD33’. and CLL-1 CD33 ) were seeded into the 96-well solid white round bottom plates with three replicated wells for each group. CAR-T (e.g., dual-CAR) T cells at various Effector: Target (E : T) ratios were seeded into the same 96-well plates. The culture medium for the co-culture as well as effector 1:2 serial dilution was IL-2-free and phenol-red free, 10% FBS- supplemented Gibco™ CTS™ AIM-V™ Medium. A group of AML cells-only samples were included as the no-killing controls to calculate the % of relative light unit (RLU).After overnight coculture (-22 hours), the assay plates were subjected to luciferase activity measurements using the lysis reagent and substrate from Promega following the manufacturer's instructions. Briefly, cells were centrifuged at 400 g for 5 min at 4 °C, and 150 pL cell supernatant per well was removed without dislodging bottom cells. Luciferase Cell Culture Lysis Reagent (CCLR; 20 pL / well) was added and cells were lysed at room temperature for 20-25 min. Then, substrate (50 pL / well) was dispensed into each well and the substrate and cell-lysed solution were mixed in wells by pipetting 2-3 times. With 10 min - 20 min after substrate addition, the RLU values of each group were obtained by reading the plates using a SpectraMax iD3 microplate reader (Molecular Devices).

[0408] The % of RLU to the control AML cells-only group is calculated to determine the potency of CAR-T cells using the following formula: % of RLU = [RLU of the (Effector + Target) group] / RLU of the Target-only group x 100%. Results are shown as the mean ± SD of three replicates from one representative donor.

[0409] Cytotoxicity assays using the xCELLigence real-time cell analysis (RTCA) system

[0410] (1) Day 0: wild type A549 cells (A549-WT) and A549 cells with antigen overexpression (CLL-1-OE, CD33-OE, and CLL-l / CD33-dOE) were seeded when the cells reached -70-90% contluency. The target cell lines were passaged and counted, and the number of cells needed was calculated. (2) 50 pL target cell medium (DMEM with 10% FBS and Pen / Strep for A549 cells) was added to all the wells on the RTCA e-plates for the Step 1 baseline read. The RTCA machine was set up according to plate layouts, and each row and column were labeled accordingly.

[0411] (3) After baseline reading, 50 pL / well of A549 target cells (2xl0e4 cells / well) were added to the appropriate rows / columns on the e-plates with a multichannel pipette and reservoirs. The e-plates were incubated in the biological safety cabinet for -30 mins to equilibrate at room temperature. The e-plates were placed back in the RTCA incubator reader to start Step 2 (1000 sweeps. 15 min interval).

[0412] (4) Day 1 (-24 hours after seeding A549 cells in e-plates): T cells were harvested and 1:2 serial dilutions were performed in AIM V basal medium (with 10% FBS and Pen / Strep) to get T cells for desired concentrations. The T cells were plated in the RTCA e-plates with specified E:T ratios (8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1). 100 pL of AIM V basal medium was added to the wells with target cells only.(5) The e-plates were placed back in the RTCA incubator reader and Step 2 of the RTCA program resumed. The RTCA instrument was allowed to monitor impedance for a desired duration with readings taken every 15 min.

[0413] (6) After releasing the RTCA running, result analysis was performed and RTCA data were exported into an Excel file with the information of parameters including normalized Cell Index, growth graph, and % of Cytolysis. The % of Cytolysis is calculated using the following formula: % of Cytolysis = [Normalized Cell Index of the Target only group - Normalized Cell Index of the (Effector + Target) group] / Normalized Cell Index of the Target only group x 100%. Results were graphed with the GraphPad Prism 9 software and shown as the mean ± SD of three replicates.

[0414] VHH antibody production

[0415] (1) Alpaca immunization. Adult alpacas were subcutaneously immunized with recombinant human CLL-l-His (His65-Ala265, UniProt Accession No. Q5QGZ9) protein or recombinant human CD33-hFc or CD33-His (Aspl8-His259, UniProt Accession No. P20138) proteins (involved Freund's complete adjuvant and Freund's incomplete adjuvant) once every other week or every week for 3-6 times. Alpaca blood was collected 7 days after the third to sixth immunizations and used for the determination of antibody titer and PBMCs isolation.

[0416] (2) Serum titer determination. Serum antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). Briefly, diluted human CLL-l-His protein or CD33-His protein was used as an antigen to coat a 96-well plate. After blocking, gradient-diluted serum was applied to the wells, and the plate was incubated at room temperature for 2 hours. After washing the plate, horseradish peroxidase (HRP)-conjugated goat anti-alpaca IgG (H+L) (Jackson / 128-035-160; 1:10000 dilution) or rabbit anti-camelid VHH (GenScript / A01861; 1:10000 dilution) was added. After incubation and washing, the TMB color developing solution was added. Further, the termination solution was added, and OD450nm was obtained.

[0417] (3) Amplification of VHH antibody sequences. Lymphocytes from immunized alpacas were extracted using a camel peripheral blood lymphocyte extraction kit according to the manufacturer's instructions. The total RNA of lymphocytes was extracted using an RNA extraction kit, and the cDNA was prepared using a reverse transcription kit. VHHs from cDNA were amplified by two-round PCR: the first round was performed using the total cDNA as templates, while the second round used the 700-bp products of the first round PCR (recovered from 1% agarose gelelectrophoresis) as templates.

[0418] (4) VHH phage library generation. Amplified VHHs were inserted into phagemids and further transduced into E. coli via electroporation. The transformed cells were cultured overnight on LB solid medium. Then, 48 single clones were randomly selected for bacterial liquid PCR and subsequent sequencing. Library size (VHH capacity 10e8 - 10e9) and insertion ratios (>90%) were determined after sequencing and verified to meet the requirements of library diversity.

[0419] (5) Phage display panning and screening. A total of 3 rounds of solid-phase panning were performed to enrich positive clones and increase phage titer. Monoclonal ELISA screening was used for solid-phase panning. Briefly, a 96-well plate was coated with the recombinant human CLL-l-His or CD33-His protein, and the analyte and the secondary antibody (HRP-conjugated mouse anti-FLAG, FLAG is the tag protein of phagemid vector; Sigma, A8592 / SLCF0816, 1:10000 dilution) were added sequentially after appropriate plate washing and blocking. The wells with secondary antibody only (in the absence of monoclonal supernatant) were considered as a blank group. After color development with TMB reagent, the OD450nm value was measured. The phage clones with the qualified OD450nm value (> 3 folds of blank) were selected for amplification, plasmid extraction and sequencing.

[0420] (6) VHH antibody characterization. The unique clones of selected VHHs were subjected to gene synthesis, subcloning, plasmid preparation, transient expression and protein purification. The purified VHH antibodies (SDS-PAGE >95%, endotoxin level <lEU / mg) were further characterized by ELISA to determine the EC50 (half-maximal effective concentration) and by Surface Plasmon Resonance (SPR) assay to determine VHH binding affinity (KD).

[0421] For ELISA, human IgGl-Fc linked VHHs (VHH-hFc) at various concentrations were incubated with plate-bound His-tagged recombinant antigen protein (CLL-l-His or CD33-His). HRP-conjugated anti-human IgG-Fc 2nd antibody was applied and OD450nm values were obtained to calculate EC50.

[0422] For SPR, VHH-hFc proteins at 1 pg / mL or 2 pg / mL were used as the capture ligand (10 pL / min, capture about 386-569 RU for anti-CLL-1 VHH and 238-862 RU for anti-CD33 VHH). The association / dissociation flow rate was set as 30 pL / min with association for 100 sec and dissociation for 120-200 sec, while the regeneration flow rate was 30 pL / min for 30 sec. The Glycine-HCl buffer (pH1.5. lOmM) was used for washing.SEQUENCES

[0423] (I) Common domains of CAR (III) Anti-CLL-1 mono- constructs (II) Anti-CLL-1 VHHs CARs

[0424] CD8 signal peptide (SP) CLL-A01 VHH CLL-A01 CAR

[0425] CD8 hinge (H) domain CLL-A02 VHH CLL-A02 CAR

[0426] CD8 transmembrane (TM) domain CLL-A03 VHH CLL-A03 CAR

[0427] 4- IBB co-stimulatory region CLL-A04 VHH CLL-A04 CAR

[0428] CD3z cytoplasmic signaling domain CLL-A05 VHH CLL-A05 CAR

[0429] P2A peptide CLL-A06 VHH CLL-A06 CAR

[0430] CD28 signal peptide (SP) CLL-A07 VHH CLL-A07 CAR

[0431] CD28 hinge (H) domain CLL-A08 VHH CLL-A08 CAR

[0432] CD28 transmembrane (TM) domain CLL-A09 VHH CLL-A09 CAR

[0433] CLL-A10 VHH CLL-A10 CAR

[0434] CLL-A11 VHH CLL-A11 CAR

[0435] CLL-A12 VHH CLL-A12 CAR

[0436] (V) Anti-CD33 mono- (VI) AxBy-28HTM dual- (IV) Anti-CD33 VHHs CARs CARs

[0437] CD33-B01 VHH CD33-B01 CAR A04B04-28HTM dual-CAR CD33-B02 VHH CD33-B02 CAR A04B06-28HTM dual-CAR CD33-B03 VHH CD33-B03 CAR A04B11-28HTM dual-CAR CD33-B04 VHH CD33-B04 CAR A07B04-28HTM dual-CAR CD33-B05 VHH CD33-B05 CAR A07B06-28HTM dual-CAR CD33-B06 VHH CD33-B06 CAR A07B11-28HTM dual-CAR CD33-B07 VHH CD33-B07 CAR A09B04-28HTM dual-CAR CD33-B08 VHH CD33-B08 CAR A09B06-28HTM dual-CAR CD33-B09 VHH CD33-B09 CAR A09B11-28HTM dual-CAR A07B11-28HTM-V3.2 dual- CAR CD33-B10 VHH CD33-B10 CAR

[0438] (codon-optimized version of A07B11-28HTM)

[0439]

[0440] CD33-B11 VHH CD33-B11 CAR

[0441] (I) Common domains of CAR constructs

[0442] CD8 signal peptide (SP) - Amino Acids (SEQ ID NO:1):

[0443] MALPVTALLLPLALLLHAARP (21 aa)

[0444] CD8 signal peptide (SP) - Nucleotides (SEQ ID NO:2):ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (63 bp)

[0445] CD8 hinge (H) domain - Amino Acids (SEQ ID NO:3):

[0446] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (55 aa)

[0447] CD8 hinge (H) domain - Nucleotides (SEQ ID NO:4):

[0448] TTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGC GCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGG CGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (165 bp)

[0449] CD8 transmembrane (TM) domain - Amino Acids (SEQ ID NO:5):

[0450] IYIWAPLAGTCGVLLLSLVITLYC (24 aa)

[0451] CD8 transmembrane (TM) domain - Nucleotides (SEQ ID NO:6):

[0452] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTAC TGC (72 bp)

[0453] 4-1BB co-stimulatory region - Amino Acids (SEQ ID NO:7):

[0454] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (42 aa)

[0455] 4-1BB co-stimulatory region - Nucleotides (SEQ ID NO:8):

[0456] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAA GAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (126 bp)

[0457] CD3zeta cytoplasmic signaling domain - Amino Acids (SEQ ID NO:9):

[0458] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (113 aa)

[0459] CD3zeta cytoplasmic signaling domain - Nucleotides (SEQ ID NO:10):

[0460] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAG ATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGAT AAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGA TGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTG CCCCCTCGC (339 bp)

[0461] P2A peptide -Amino Acids (SEQ ID NO:11):

[0462] GSGATNFSLLKQAGDVEENPGP (22aa)

[0463] P2A peptide - Nucleotides (SEQ ID NO:12):

[0464] GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT

[0465] (66 bp)

[0466] CD28 signal peptide (SP) - Amino Acids (SEQ ID NO:13):

[0467] MLRLLLALNLFPSIQVTG (18 aa)

[0468] CD28 signal peptide (SP) - Nucleotides (SEQ ID NO: 14):

[0469] ATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTTCAATTCAAGTAACAGGA (54 bp)

[0470] CD28 hinge (H) domain - Amino Acids (SEQ ID NO:15):

[0471] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (39 aa)

[0472] CD28 hinge (H) domain - Nucleotides (SEQ ID NO:16):

[0473] ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAG GGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (117 bp)

[0474] CD28 transmembrane (TM) domain - Amino Acids (SEQ ID NO:17):

[0475] FWVLVVVGGVLACYSLLVTVAFIIFWV (27 aa)

[0476] CD28 transmembrane (TM) domain - Nucleotides (SEQ ID NO: 18):

[0477] TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTA11299 / 013170-WG0

[0478] TTTTCTGGGTG (81 bp)

[0479] (II) Anti-CLL-1 VHHs

[0480] CLL-A01 VHH - Amino Acids (SEQ ID NO:19):

[0481] AVQLVESGGGLVQVGGSLRLSCVASGRTFSSYITMGWFRQAPGKEREFVASIIGNGGFTYYAASVKGRFTIS RENAKNSLYLQMDDLKPEDTAIYYCAADRGWTRALGARGDEYEDWGQGTQVTVSS (127 aa)

[0482] CLL-AO1 VHH - Nucleotides (SEQ ID NO:20):

[0483] GCTGTTCAGTTGGTGGAGTCCGGAGGAGGATTGGTGCAGGTGGGAGGATCTTTGAGGTTGAGTTGCGTTG CTTCTGGGAGGACTTTTTCTTCTTATATTACTATGGGATGGTTCAGGCAGGCTCCTGGGAAGGAGAGGGA GTTTGTGGCTTCTATCATTGGAAACGGCGGGTTCACCTACTACGCTGCCAGCGTTAAGGGCAGGTTCACT ATCTCCCGGGAGAACGCCAAGAACAGCTTGTATCTGCAGATGGACGATCTCAAGCCCGAGGACACCGCTA TCTATTACTGCGCCGCCGACAGGGGATGGACCAGAGCTTTGGGTGCCAGGGGTGACGAGTACGAAGACTG GGGACAGGGGACCCAGGTGACAGTTTCCTCC (381 bp)

[0484] CLL-A01 VHH

[0485] Residues SEQ of SEQ ID NO Region Amino Acid Sequence Length

[0486] ID NO: 19

[0487] SEQ FR1 AVQLVESGGGLVQVGGSLRLSCVASGRTFS 1 - 30 30 ID NO:21 SEQ CDR1 SYITMG 31 - 36 6 ID NO:22 SEQ FR2 WFRQAPGKEREFVA 37 - 50 14 ID NO:23 SEQ CDR2 SIIGNGGFTYYAASVKG 51 - 67 17 ID NO:24 SEQ FR3 RFTISRENAKNSLYLQMDDLKPEDTAIYYCAA 68 - 99 32 ID NO:25 SEQ

[0488] 100 - CDR3 DRGWTRALGARGDEYED 17 ID

[0489] 116

[0490]

[0491] NO:26SEQ 117 - FR4 WGQGTQVTVSS 11 ID 127

[0492] NO:27

[0493]

[0494] 127

[0495] CLL-A01 VHH

[0496] Region DNA Sequence SEQ ID NO FR1 GCTGTTCAGTTGGTGGAGTCCGGAGGAGGATTGGTGCA SEQ ID NO:28

[0497] GGTGGGAGGATCTTTGAGGTTGAGTTGCGTTGCTTCTG GGAGGACTTTTTCT CDR1 TCTTATATTACTATGGGA SEQ ID NO:29 FR2 TGGTTCAGGCAGGCTCCTGGGAAGGAGAGGGAGTTTG SEQ ID NO:30

[0498] TGGCT CDR2 TCTATCATTGGAAACGGCGGGTTCACCTACTACGCTGCC SEQ ID NO:31

[0499] AGCGTTAAGGGC FR3 AGGTTCACTATCTCCCGGGAGAACGCCAAGAACAGCTT SEQ ID NO:32

[0500] GTATCTGCAGATGGACGATCTCAAGCCCGAGGACACCG CTATCTATTACTGCGCCGCC CDR3 GACAGGGGATGGACCAGAGCTTTGGGTGCCAGGGGTG SEQ ID NO:33

[0501] ACGAGTACGAAGAC FR4 TGGGGACAGGGGACCCAGGTGACAGTTTCCTCC SEQ ID NO:34

[0502]

[0503] CLL-A02 VHH - Amino Acids (SEQ ID NO:35):

[0504] QLQLVESGGGLVQAGGSLRLSCAASGRTFSNYIMGWFRQAPGKEREFVAAITWSGGSTYYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCAARDDPLLLLRLWMLWNEYDYWGQGTQVTVSS (128 aa)

[0505] CLL-A02 VHH - Nucleotides (SEQ ID NO:36):

[0506] CAGTTGCAGCTCGTGGAGTCCGGAGGAGGACTCGTTCAGGCAGGAGGATCACTCAGGCTCTCCTGCGCTG CTTCCGGAAGAACCTTCTCCAACTACATCATGGGCTGGTTCAGGCAGGCCCCCGGAAAAGAAAGGGAGTT CGTGGCTGCCATCACCTGGAGCGGAGGTTCTACCTACTACGCCGACTCCGTCAAGGGCAGATTCACAATC AGCAGGGACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCTTGAAGCCCGAGGACACCGCCGTGT ACTACTGCGCTGCTAGGGATGACCCCCTGCTGTTGCTGCGTCTGTGGATGCTGTGGAACGAGTACGACTA CTGGGGCCAGGGCACCCAGGTTACTGTGTCTAGT (384 bp)CLL-A02 VHH

[0507] Region Amino Acid Sequence Residues of Length SEQ SEQ ID ID NO NO:35

[0508] FR1 QLQLVESGGGLVQAGGSLRLSCAASGRTFS 1 - 30 30 SEQ ID NO:37 CDR1 NYIMG 31 - 35 5 SEQ ID NO:38 FR2 WFRQAPGKEREFVA 36 - 49 14 SEQ ID NO:39 CDR2 AITWSGGSTYYADSVKG 50 - 66 17 SEQ ID NO:40 FR3 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCA 67 - 98 32 SEQ A ID NO:41 CDR3 RDDPLLLLRLWMLWNEYDY 99 - 117 19 SEQ ID NO:42 FR4 WGQGTQVTVSS 118 - 128 11 SEQ ID NO:43 128

[0509]

[0510] CLL-A02 VHH

[0511] Region DNA sequence SEQ ID NO FR1 CAGTTGCAGCTCGTGGAGTCCGGAGGAGGACTCGTTCA SEQ ID NO:44

[0512] GGCAGGAGGATCACTCAGGCTCTCCTGCGCTGCTTCCGG AAGAACCTTCTCC CDR1 AACTACATCATGGGC SEQ ID NO:45 FR2 TGGTTCAGGCAGGCCCCCGGAAAAGAAAGGGAGTTCGT SEQ ID NO:46

[0513]

[0514] GGCTCDR2 GCCATCACCTGGAGCGGAGGTTCTACCTACTACGCCGAC SEQ ID NO:47 TCCGTCAAGGGC FR3 AGATTCACAATCAGCAGGGACAACGCCAAGAACACCGT SEQ ID NO:48

[0515] GTACCTGCAGATGAACAGCTTGAAGCCCGAGGACACCG CCGTGTACTACTGCGCTGCT CDR3 AGGGATGACCCCCTGCTGTTGCTGCGTCTGTGGATGCTG SEQ ID NO:49

[0516] TGGAACGAGTACGACTAC

[0517]

[0518] FR4 TGGGGCCAGGGCACCCAGGTTACTGTGTCTAGT SEQ ID NO:50 CLL-A03 VHH - Amino Acids (SEQ ID NO:51):

[0519] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEHEFVAAISWSGGSTYYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCAARDGGFYDSDWRRSRNEYDYWGQGTQVTVSS (128 aa)

[0520] CLL-A03 VHH - Nucleotides (SEQ ID NO:52):

[0521] GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTGCAGGCAGGAGGAAGCCTGAGGCTGAGTTGTGCCG CTAGCGGAAGGACATTTAGTAGCTACGCCATGGGATGGTTTAGGCAGGCCCCAGGAAAGGAGCACGAGTT CGTGGCTGCAATTTCCTGGAGCGGAGGAAGCACATATTACGCCGATTCTGTGAAGGGCAGGTTCACCATC TCCCGGGACAACGCTAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCCGAAGACACCGCCGTGT ACTACTGCGCTGCCAGAGACGGAGGATTCTACGACTCCGACTGGAGGAGGAGCAGAAACGAGTACGACTA CTGGGGCCAGGGAACCCAGGTGACAGTGTCTAGC (384 bp)

[0522] CLL-A03 VHH

[0523] Region Amino Acid Sequence Residues Length SEQ of SEQ ID NO ID NO:51

[0524] FR1 EVQLVESGGGLVQAGGSLRLSCAASGRTFS 1 - 30 30 SEQ ID NO:53 CDR1 SYAMG 31 - 35 5 SEQ ID NO:54 FR2 WFRQAPGKEHEFVA 36 - 49 14 SEQ ID NO:55 CDR2 AISWSGGSTYYADSVKG 50 - 66 17 SEQ ID NO:56 FR3 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA 67 - 98 32 SEQ

[0525] ID

[0526]

[0527] NO:57CDR3 RDGGFYDSDWRRSRNEYDY 99 - 117 19 SEQ ID NO:58 FR4 WGQGTQVTVSS 118 - 11 SEQ 128 ID

[0528] NO:59

[0529]

[0530] 128

[0531] CLL-A03 VHH

[0532] Region DNA sequence SEQ ID NO FR1 GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTG SEQ ID CAGGCAGGAGGAAGCCTGAGGCTGAGTTGTGCCGCT NO:60 AGCGGAAGGACATTTAGT CDR1 AGCTACGCCATGGGA SEQ ID NO:61

[0533] FR2 TGGTTTAGGCAGGCCCCAGGAAAGGAGCACGAGTTC SEQ ID GTGGCT NO:62 CDR2 GCAATTTCCTGGAGCGGAGGAAGCACATATTACGCCG SEQ ID ATTCTGTGAAGGGC NO:63 FR3 AGGTTCACCATCTCCCGGGACAACGCTAAGAACACCG SEQ ID TGTACCTGCAGATGAACAGCCTGAAGCCCGAAGACA NO:64 CCGCCGTGTACTACTGCGCTGCC CDR3 AGAGACGGAGGATTCTACGACTCCGACTGGAGGAGG SEQ ID AGCAGAAACGAGTACGACTAC NO:65 FR4 TGGGGCCAGGGAACCCAGGTGACAGTGTCTAGC SEQ ID

[0534] NO:66

[0535]

[0536] CLL-A04 VHH - Amino Acids (SEQ ID NO:67):

[0537] EVQLVESGGGLVQVGGSLRLSCVASGRTFSSYITMGWFRQAPGKEREFVASIIGNDAFTYYAASVKGRFTISR ENAKNSLYLQMDDLKPEDTATYFCAADRGWTAALGARGDHYEDWGQGTQVTVSS (127 aa)

[0538] CLL-A04 VHH - Nucleotides (SEQ ID NO:68):

[0539] GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTGCAGGTGGGAGGAAGCCTGAGGCTGAGTTGTGTGG CTAGCGGGAGGACATTTAGTTCCTATATCACAATGGGGTGGTTCAGGCAGGCTCCAGGAAAGGAGAGGGA GTTCGTGGCTAGTATTATTGGAAACGACGCCTTCACCTACTACGCTGCCTCAGTGAAGGGCCGGTTCACC ATCAGCAGGGAGAATGCTAAGAACAGCCTGTACCTGCAGATGGATGATTTGAAGCCCGAGGACACAGCTA CCTACTTCTGCGCCGCCGACCGAGGATGGACAGCTGCTTTGGGAGCACGGGGAGACCACTACGAGGACTGGGGACAGGGAACACAGGTGACCGTGAGCTCC (381 bp)

[0540] CLL-A04VHH

[0541] Residues SEQ ID of SEQ NO Region Amino Acid Sequence Length

[0542] ID NO:67

[0543] FR1 SEQ ID EVQLVESGGGLVQVGGSLRLSCVASGRTFS 1 - 30 30 NO:69 CDR1 SEQ ID SYITMG 31 - 36 6

[0544] NO:70 FR2 SEQ ID WFRQAPGKEREFVA 37 - 50 14

[0545] NO:71 CDR2 SEQ ID SIIGNDAFTYYAASVKG 51 - 67 17

[0546] NO:72 FR3 SEQ ID RFTISRENAKNSLYLQMDDLKPEDTATYFCAA 68 - 99 32

[0547] NO:73 CDR3 100 - SEQ ID DRGWTAALGARGDHYED 17

[0548] 116 NO:74 FR4 117 - SEQ ID WGQGTQVTVSS 11

[0549] 127 NO:75

[0550]

[0551] 127

[0552] CLL-A04 VHH

[0553] Region DNA sequence SEQ ID NO FR1 GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTGCA SEQ ID NO:76

[0554] GGTGGGAGGAAGCCTGAGGCTGAGTTGTGTGGCTAGCG GGAGGACATTTAGT CDR1 TCCTATATCACAATGGGG SEQ ID NO:77 FR2 TGGTTCAGGCAGGCTCCAGGAAAGGAGAGGGAGTTCGT SEQ ID NO:78

[0555] GGCT CDR2 AGTATTATTGGAAACGACGCCTTCACCTACTACGCTGCCT SEQ ID NO:79

[0556] CAGTGAAGGGC FR3 CGGTTCACCATCAGCAGGGAGAATGCTAAGAACAGCCTG SEQ ID NO:80

[0557] TACCTGCAGATGGATGATTTGAAGCCCGAGGACACAGCT ACCTACTTCTGCGCCGCC CDR3 GACCGAGGATGGACAGCTGCTTTGGGAGCACGGGGAGA SEQ ID NO:81

[0558] CCACTACGAGGAC

[0559]

[0560] FR4 TGGGGACAGGGAACACAGGTGACCGTGAGCTCC SEQ ID NO:82

[0561] CLL-A05 VHH - Amino Acids (SEQ ID NO:83):

[0562] QLHFVESGGGLVQAGGSLRLSCAASGRTFSSYITMGWFRQAPGKEREFVASIIGNGGFTYYAASVKGRFTIS RDNAKNSLYLQMDSLKPEDTAVYYCAADRGWTADLGTRGDDYEDWGQGTQVTVSS (127 aa)CLL-A05 VHH - Nucleotides (SEQ ID NO:84):

[0563] CAGTTGCACTTCGTGGAGTCCGGCGGAGGACTCGTTCAGGCTGGAGGATCACTCAGGCTCTCCTGCGCTG CTTCCGGAAGAACCTTCTCCTCCTACATCACCATGGGCTGGTTCAGGCAGGCCCCTGGAAAGGAACGCGA GTTTGTGGCCAGTATCATTGGAAACGGGGGCTTCACTTACTACGCCGCCTCCGTTAAGGGCAGGTTCACC ATCTCCCGGGACAACGCTAAGAACAGCCTGTACTTGCAGATGGACAGCCTGAAGCCTGAGGACACCGCTG TGTACTACTGTGCAGCCGACAGAGGATGGACCGCAGATCTGGGTACAAGGGGCGACGACTACGAAGACTG GGGACAGGGAACCCAGGTGACCGTTTCCTCC (381 bp)

[0564] CLL-A05 VHH

[0565] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO:83

[0566] FR1 QLHFVESGGGLVQAGGSLRLSCAASGRTFS 1 - 30 30 SEQ ID NO:85 CDR1 SYITMG 31 - 36 6 SEQ ID NO:86 FR2 WFRQAPGKEREFVA 37 - 50 14 SEQ ID NO:87 CDR2 SIIGNGGFTYYAASVKG 51 - 67 17 SEQ ID NO:88 FR3 RFTISRDNAKNSLYLQMDSLKPEDTAVYYCAA 68 - 99 32 SEQ ID NO:89 CDR3 DRGWTADLGTRGDDYED 100 - 17 SEQ ID 116 NO:90 FR4 WGQGTQVTVSS 117 - 11 SEQ ID 127 NO:91

[0567]

[0568] 127

[0569] CLL-A05 VHH

[0570] Region DNA sequence SEQ ID NO FR1 CAGTTGCACTTCGTGGAGTCCGGCGGAGGACTCGTTC SEQ ID NO:92

[0571] AGGCTGGAGGATCACTCAGGCTCTCCTGCGCTGCTTCC GGAAGAACCTTCTCC

[0572]

[0573] CDR1 TCCTACATCACCATGGGC SEQ ID NO:93FR2 TGGTTCAGGCAGGCCCCTGGAAAGGAACGCGAGTTTG SEQ ID NO:94 TGGCC CDR2 AGTATCATTGGAAACGGGGGCTTCACTTACTACGCCGC SEQ ID NO:95 CTCCGTTAAGGGC FR3 AGGTTCACCATCTCCCGGGACAACGCTAAGAACAGCC SEQ ID NO:96 TGTACTTGCAGATGGACAGCCTGAAGCCTGAGGACAC CGCTGTGTACTACTGTGCAGCC CDR3 GACAGAGGATGGACCGCAGATCTGGGTACAAGGGGCG SEQ ID NO:97

[0574] ACGACTACGAAGAC

[0575]

[0576] FR4 TGGGGACAGGGAACCCAGGTGACCGTTTCCTCC SEQ ID NO:98 CLL-A06 VHH - Amino Acids (SEQ ID NO:99):

[0577] ELQLVESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISR DNAKNTVYLQMNSLKVEDTAVYYCNEVPPYGTSWYAEDEYWGQGTQVTVSS (122 aa)

[0578] CLL-A06 VHH - Nucleotides (SEQ ID N0:100):

[0579] GAACTGCAGCTGGTGGAATCCGGCGGAGGATTGGTGCAGCCTGGAGGATCTTTGAGGCTGAGCTGCGCCG CTTCCGGAAGTATCTTTAGTATTAACGCCATGGGATGGTACAGGCAGGCCCCTGGAAAGCAGAGGGAGTT GGTGGCTGCTATTACCTCCGGAGGAAGCACCAACTACGCAGACAGCGTGAAGGGGAGGTTCACCATCAGC AGGGATAACGCAAAGAACACCGTCTACCTGCAGATGAACAGCCTGAAGGTGGAAGATACCGCAGTGTACT ACTGCAACGAAGTGCCCCCCTACGGCACAAGTTGGTACGCAGAGGACGAGTACTGGGGACAGGGCACACA GGTGACCGTGAGTTCC (366 bp)

[0580] CLL-A06 VHH

[0581] Region Sequence Fragment Residues Length SEQ ID of SEQ NO ID NO:99

[0582] FR1 ELQLVESGGGLVQPGGSLRLSCAASGSIFS 1 - 30 30 SEQ ID NO:101 CDR1 INAMG 31 - 35 5 SEQ ID NO: 102 FR2 WYRQAPGKQRELVA 36 - 49 14 SEQ ID NO: 103 CDR2 AITSGGSTNYADSVKG 50 - 65 16 SEQ ID NO: 104

[0583]

[0584] FR3 RFTISRDNAKNTVYLQMNSLKVEDTAVYYCNE 66 - 97 32 SEQ ID NO:105 CDR3 VPPYGTSWYAEDEY 98 - 111 14 SEQ ID NO: 106 FR4 WGQGTQVTVSS 112 - 11 SEQ ID 122 NO: 107

[0585]

[0586] 122

[0587] CLL-A06 VHH

[0588] Region DNA sequence SEQ ID NO FR1 GAACTGCAGCTGGTGGAATCCGGCGGAGGATTGGTGCAGCCT SEQ ID GGAGGATCTTTGAGGCTGAGCTGCGCCGCTTCCGGAAGTATC NO:108 TTTAGT CDR1 ATTAACGCCATGGGA SEQ ID NO:109 FR2 TGGTACAGGCAGGCCCCTGGAAAGCAGAGGGAGTTGGTGGC SEQ ID T NO:110 CDR2 GCTATTACCTCCGGAGGAAGCACCAACTACGCAGACAGCGTG SEQ ID AAGGGG NO:111 FR3 AGGTTCACCATCAGCAGGGATAACGCAAAGAACACCGTCTAC SEQ ID CTGCAGATGAACAGCCTGAAGGTGGAAGATACCGCAGTGTAC NO:112 TACTGCAACGAA CDR3 GTGCCCCCCTACGGCACAAGTTGGTACGCAGAGGACGAGTAC SEQ ID NO:113 FR4 TGGGGACAGGGCACACAGGTGACCGTGAGTTCC SEQ ID

[0589]

[0590] NO:114 CLL-A07 VHH - Amino Adds (SEQ ID NO:115):

[0591] AVQLVESGGGLVQPGGSLRLFCAASGFTFGSYDMSWVRQAPGKEVEWVSTINSRGDRTYYPDSVKGRFTI SRDNAKNTVSLQMNNLKPEDTAVYYCATQETPGGDEAYWGQGTQVTVSS (119 aa)

[0592] CLL-A07 VHH - Nudeotides (SEQ ID NO:116):

[0593] GCTGTTCAGTTGGTGGAGTCCGGAGGAGGATTGGTGCAGCCTGGTGGATCTTTGAGGTTGTTTTGTGCTG CTTCTGGGTTTACCTTTGGATCTTATGATATGTCCTGGGTGAGGCAGGCCCCCGGAAAAGAAGTGGAGTG GGTGAGCACTATTAACTCCAGGGGGGACAGGACATACTACCCCGATTCCGTTAAGGGCCGGTTTACAATC TCCCGTGACAACGCTAAGAACACCGTGAGCCTGCAGATGAATAACTTGAAGCCCGAGGACACCGCCGTGT ACTACTGTGCTACCCAGGAAACCCCCGGCGGAGATGAAGCCTATTGGGGACAGGGAACACAGGTGACCGT GAGTTCC (357 bp)CLL-A07 VHH

[0594] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO:115

[0595] FR1 AVQLVESGGGLVQPGGSLRLFCAASGFTFG 1 - 30 30 SEQ ID NO:117 CDR1 SYDMS 31 - 35 5 SEQ ID NO:118 FR2 WVRQAPGKEVEWVS 36 - 49 14 SEQ ID NO:119 CDR2 TINSRGDRTYYPDSVKG 50 - 66 17 SEQ ID NO: 120 FR3 RFTISRDNAKNTVSLQMNNLKPEDTAVYYCAT 67 - 98 32 SEQ ID NO:121 CDR3 QETPGGDEAY 99 - 108 10 SEQ ID NO: 122 FR4 WGQGTQVTVSS 109 - 11 SEQ ID 119 NO: 123

[0596]

[0597] 119

[0598] CLL-A07 VHH

[0599] Region DNA sequence SEQ ID NO FR1 GCTGTTCAGTTGGTGGAGTCCGGAGGAGGATTGGTGCAGC SEQ ID CTGGTGGATCTTTGAGGTTGTTTTGTGCTGCTTCTGGGTTT NO: 124 ACCTTTGGA CDR1 TCTTATGATATGTCC SEQ ID NO: 125 FR2 TGGGTGAGGCAGGCCCCCGGAAAAGAAGTGGAGTGGGTG SEQ ID AGC NO: 126 CDR2 ACTATTAACTCCAGGGGGGACAGGACATACTACCCCGATTC SEQ ID CGTTAAGGGC NO: 127

[0600]

[0601] FR3 CGGTTTACAATCTCCCGTGACAACGCTAAGAACACCGTGA SEQ ID GCCTGCAGATGAATAACTTGAAGCCCGAGGACACCGCCGT NO:128 GTACTACTGTGCTACC CDR3 CAGGAAACCCCCGGCGGAGATGAAGCCTAT SEQ ID NO: 129 FR4 TGGGGACAGGGAACACAGGTGACCGTGAGTTCC SEQ ID

[0602] NO:130

[0603]

[0604] CLL-A08 VHH - Amino Acids (SEQ ID NO:131):

[0605] EVHLVESGEGLVQVGGSLRLSCVASGRTFSSYITMGWFRQAPGKEREFVASIIGNGGFTYYAASVKGRFTISR ENAKNSLYLQMDDLKPEDTAIYYCAADRGWTAALGARGDDYEDWGQGTQVTVSS (127 aa)

[0606] CLL-A08 VHH - Nucleotides (SEQ ID NO:132):

[0607] GAAGTGCACCTGGTGGAGTCCGGAGAGGGATTGGTGCAGGTGGGAGGAAGCCTGAGGCTGAGTTGTGTGG CCAGTGGCAGGACATTTAGCAGCTACATCACAATGGGGTGGTTTAGGCAGGCCCCCGGAAAGGAAAGGGA GTTTGTGGCTAGTATTATCGGAAATGGCGGCTTCACTTACTACGCCGCATCCGTTAAGGGCAGGTTCACC ATCAGCAGGGAGAACGCCAAGAACAGCCTGTACTTGCAGATGGACGACCTGAAGCCTGAGGACACCGCTA TCTATTACTGCGCAGCCGACAGGGGATGGACCGCTGCTTTGGGAGCAAGGGGAGACGACTACGAAGACTG GGGACAGGGCACCCAGGTGACAGTTTCCTCC (381 bp)

[0608] CLL-A08 VHH

[0609] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO:131

[0610] FR1 EVHLVESGEGLVQVGGSLRLSCVASGRTFS 1 - 30 30 SEQ ID NO:133 CDR1 SYITMG 31 - 36 6 SEQ ID NO:I34 FR2 WFRQAPGKEREFVA 37 - 50 14 SEQ ID NO:135 CDR2 SIIGNGGFTYYAASVKG 51 - 67 17 SEQ ID NO:136 FR3 RFTISRENAKNSLYLQMDDLKPEDTAIYYCAA 68 - 99 32 SEQ ID

[0611] NO:137

[0612]

[0613] CDR3 DRGWTAALGARGDDYED 100 - 17 SEQ ID 116 NO:138 FR4 WGQGTQVTVSS 117 - 11 SEQ ID 127 NO:139

[0614]

[0615] 127

[0616] CLL-A08 VHH

[0617] Region DNA sequence SEQ ID NO FR1 GAAGTGCACCTGGTGGAGTCCGGAGAGGGATTGGTGCAGGTG SEQ ID GGAGGAAGCCTGAGGCTGAGTTGTGTGGCCAGTGGCAGGACA NO: 140 TTTAGC CDR1 AGCTACATCACAATGGGG SEQ ID NO:141 FR2 TGGTTTAGGCAGGCCCCCGGAAAGGAAAGGGAGTTTGTGGCT SEQ ID NO: 142 CDR2 AGTATTATCGGAAATGGCGGCTTCACTTACTACGCCGCATCCGT SEQ ID TAAGGGC NO: 143 FR3 AGGTTCACCATCAGCAGGGAGAACGCCAAGAACAGCCTGTAC SEQ ID TTGCAGATGGACGACCTGAAGCCTGAGGACACCGCTATCTATT NO: 144 ACTGCGCAGCC CDR3 GACAGGGGATGGACCGCTGCTTTGGGAGCAAGGGGAGACGAC SEQ ID TACGAAGAC NO: 145 FR4 TGGGGACAGGGCACCCAGGTGACAGTTTCCTCC SEQ ID NO: 146

[0618]

[0619] CLL-A09 VHH - Amino Adds (SEQ ID NO:147):

[0620] QVQLVESGGGLVQPGGSLRLSCAASGRTFSMDAMGWFRQAPGKEREFVALISWKSDSTFYADSVKGRFTI SRDNAKNTVSLQMNSLKPEDTAVYYCAVGYIATMSQGTMDYWGQGTQVTVSS (122 aa)

[0621] CLL-A09 VHH - Nudeotides (SEQ ID NO:148):

[0622] CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGCCAGGAGGATCACTCAGGCTGTCATGCGCCG CTTCCGGAAGAACATTCTCCATGGACGCAATGGGCTGGTTCAGGCAGGCTCCTGGAAAGGAGAGGGAGTT CGTGGCACTGATTTCCTGGAAGTCCGACTCCACATTCTACGCTGATAGTGTGAAAGGACGGTTCACCATC TCCCGGGACAATGCTAAGAACACCGTGAGCCTGCAGATGAACAGCCTGAAGCCCGAAGACACCGCCGTGTACTACTGCGCAGTTGGCTACATTGCCACCATGAGCCAGGGCACCATGGACTACTGGGGACAGGGAACCCA GGTGACCGTGTCTTCC (366 bp)

[0623] CLL-A09 VHH

[0624] Region Amino Acid Sequence Residues Length SEQ ID of SEQ No: ID NO: 147

[0625] FR1 QVQLVESGGGLVQPGGSLRLSCAASGRTFS 1 - 30 30 SEQ ID NO: 149 CDR1 MDAMG 31 - 35 5 SEQ ID NO:150 FR2 WFRQAPGKEREFVA 36 - 49 14 SEQ ID NO:151 CDR2 LISWKSDSTFYADSVKG 50 - 66 17 SEQ ID NO:152 FR3 RFTISRDNAKNTVSLQMNSLKPEDTAVYYCAV 67 - 98 32 SEQ ID NO: 153 CDR3 GYIATMSQGTMDY 99 - 111 13 SEQ ID NO: 154 FR4 WGQGTQVTVSS 112 - 11 SEQ ID 122 NO:155

[0626]

[0627] 122

[0628] CLL-A09 VHH

[0629] Region DNA sequence SEQ ID NO FR1 CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGC SEQ ID CAGGAGGATCACTCAGGCTGTCATGCGCCGCTTCCGGAAG NO:156 AACATTCTCC CDR1 ATGGACGCAATGGGC SEQ ID NO: 157 FR2 TGGTTCAGGCAGGCTCCTGGAAAGGAGAGGGAGTTCGTG SEQ ID GCA NO:158

[0630]

[0631] CDR2 CTGATTTCCTGGAAGTCCGACTCCACATTCTACGCTGATAG SEQ ID TGTGAAAGGA NO:159 FR3 CGGTTCACCATCTCCCGGGACAATGCTAAGAACACCGTGA SEQ ID GCCTGCAGATGAACAGCCTGAAGCCCGAAGACACCGCCGT NO: 160 GTACTACTGCGCAGTT CDR3 GGCTACATTGCCACCATGAGCCAGGGCACCATGGACTAC SEQ ID NO:161 FR4 TGGGGACAGGGAACCCAGGTGACCGTGTCTTCC SEQ ID NO: 162

[0632]

[0633] CLL-A10 VHH - Amino Adds (SEQ ID NO:163):

[0634] AVQLVESGGGLVQPGDSLRLFCAASGFTFGTYDMSWVRQAPGKEVEWVSTINSRGDRTYYPDSVKGRFTIS RDNAKNTLYLQMDSLKPEDTALYYCVTDAFGSWGQGTQVTVSS (114 aa)

[0635] CLL-A1O VHH - Nucleotides (SEQ ID NO:164):

[0636] GCTGTTCAGTTGGTGGAGTCCGGAGGAGGATTGGTGCAGCCTGGTGATTCCCTGAGGTTGTTTTGTGCTG CTTCTGGGTTTACCTTTGGAACTTATGATATGTCCTGGGTGAGGCAGGCCCCTGGAAAGGAAGTTGAGTG GGTGAGTACCATTAATTCCAGGGGGGACAGGACATACTACCCTGATTCCGTCAAGGGCCGCTTTACAATC TCCCGCGACAACGCTAAGAACACCCTGTACCTGCAGATGGATAGTCTGAAACCCGAGGACACAGCCTTGT ATTATTGCGTGACCGACGCCTTCGGCTCCTGGGGACAGGGAACACAGGTGACAGTGTCCTCC (342 bp)

[0637] CLL-A10 VHH

[0638] Region Sequence Fragment Residues Length SEQ ID of SEQ NO ID NO: 163

[0639] FR1 AVQLVESGGGLVQPGDSLRLFCAASGFTFG 1 - 30 30 SEQ ID NO: 165 CDR1 TYDMS 31 - 35 5 SEQ ID NO: 166 FR2 WVRQAPGKEVEWVS 36 - 49 14 SEQ ID NO: 167 CDR2 TINSRGDRTYYPDSVKG 50 - 66 17 SEQ ID

[0640] NO:168

[0641]

[0642] FR3 RFTISRDNAKNTLYLQMDSLKPEDTALYYCVT 67 - 98 32 SEQ ID NO:169 CDR3 DAFGS 99 - 103 5 SEQ ID NO:170 FR4 WGQGTQVTVSS 104- 11 SEQ ID 114 NO:171

[0643]

[0644] 114

[0645] CLL-A10 VHH

[0646] Region DNA sequence SEQ ID NO FR1 GCTGTTCAGTTGGTGGAGTCCGGAGGAGGATTGGTGCAGCCT SEQ ID GGTGATTCCCTGAGGTTGTTTTGTGCTGCTTCTGGGTTTACCT NO:172 TTGGA CDR1 ACTTATGATATGTCC SEQ ID NO:173 FR2 TGGGTGAGGCAGGCCCCTGGAAAGGAAGTTGAGTGGGTGAG SEQ ID T NO: 174 CDR2 ACCATTAATTCCAGGGGGGACAGGACATACTACCCTGATTCCG SEQ ID TCAAGGGC NO:175 FR3 CGCTTTACAATCTCCCGCGACAACGCTAAGAACACCCTGTAC SEQ ID CTGCAGATGGATAGTCTGAAACCCGAGGACACAGCCTTGTAT NO:176 TATTGCGTGACC CDR3 GACGCCTTCGGCTCC SEQ ID NO: 177 FR4 TGGGGACAGGGAACACAGGTGACAGTGTCCTCC SEQ ID

[0647] NO:178

[0648]

[0649] CLL-A11 VHH - Amino Acids (SEQ ID NO:179):

[0650] EVDLVESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGNQRELVAAITSGANTAYSDSVKGRFTISR DNAKNTVYLQMNSLKPDDTAVYYCYALIPLNGSSWPPDEYWGQGTQVTVSS (122 aa)

[0651] CLL-A11 VHH - Nucleotides (SEQ ID NO:180):

[0652] GAAGTGGACCTGGTGGAGAGCGGAGGAGGATTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGTTGTGCCG CAAGCGGAAGCATCTTTAGCATCAACGCTATGGGGTGGTACAGGCAGGCACCCGGAAACCAGAGGGAGTTGGTGGCTGCTATTACCTCTGGAGCTAACACCGCATACTCCGACAGCGTGAAGGGAAGGTTCACTATCTCC AGAGATAACGCTAAGAACACCGTCTACCTCCAGATGAACAGCCTGAAGCCTGATGATACCGCCGTTTACT ACTGCTACGCCCTGATTCCCTTGAATGGCTCCTCCTGGCCCCCCGATGAATACTGGGGACAGGGAACCCA GGTGACCGTCTCTTCC (366 bp)

[0653] CLL-A11 VHH

[0654] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO:179

[0655] FR1 EVDLVESGGGLVQPGGSLRLSCAASGSIFS 1 - 30 30 SEQ ID NO:181 CDR1 INAMG 31 - 35 5 SEQ ID NO:182 FR2 WYRQAPGNQRELVA 36 - 49 14 SEQ ID NO:183 CDR2 AITSGANTAYSDSVKG 50 - 65 16 SEQ ID NO:184 FR3 RFTISRDNAKNTVYLQMNSLKPDDTAVYYCYA 66 - 97 32 SEQ ID NO:185 CDR3 LIPLNGSSWPPDEY 98 - 111 14 SEQ ID NO:186 FR4 WGQGTQVTVSS 112 - 11 SEQ ID 122 NO:187

[0656]

[0657] 122

[0658] CLL-A11 VHH

[0659] Region DNA sequence SEQ ID NO FR1 GAAGTGGACCTGGTGGAGAGCGGAGGAGGATTGGTGCAGC SEQ ID CTGGAGGAAGCCTGAGGCTGAGTTGTGCCGCAAGCGGAAG NO:188 CATCTTTAGC CDR1 ATCAACGCTATGGGG SEQ ID

[0660] NO:189

[0661]

[0662] FR2 TGGTACAGGCAGGCACCCGGAAACCAGAGGGAGTTGGTGG SEQ ID CT NO:190 CDR2 GCTATTACCTCTGGAGCTAACACCGCATACTCCGACAGCGTG SEQ ID AAGGGA NO:191 FR3 AGGTTCACTATCTCCAGAGATAACGCTAAGAACACCGTCTAC SEQ ID CTCCAGATGAACAGCCTGAAGCCTGATGATACCGCCGTTTAC NO: 192 TACTGCTACGCC CDR3 CTGATTCCCTTGAATGGCTCCTCCTGGCCCCCCGATGAATAC SEQ ID NO: 193 FR4 TGGGGACAGGGAACCCAGGTGACCGTCTCTTCC SEQ ID NO: 194

[0663]

[0664] CLL-A12 VHH - Amino Acids (SEQ ID NO:195):

[0665] EVHLVESGGGLVQPGGSLRLSCSASGRTFSQYGVGWSRQAPGKEREIVAAIIWSDGSTYYADSVKGRFTISR DNAKNTAYLQMNSLKPEDTAVYYCAADNDLLSGLADEDADWGQGTQVTVSS (123 aa)

[0666] CLL-A12 VHH - Nucleotides (SEQ ID NO:196):

[0667] GAAGTGCACCTGGTGGAGTCCGGAGGAGGATTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGTTGTAGTG CCAGCGGAAGGACATTTTCCCAGTACGGCGTGGGGTGGAGCAGGCAGGCTCCTGGAAAAGAGAGGGAGAT TGTGGCCGCCATCATTTGGAGCGACGGGTCTACATACTACGCCGACAGTGTGAAGGGGCGGTTCACCATC AGCAGGGACAATGCCAAAAACACCGCCTACCTGCAGATGAACTCCCTGAAGCCCGAGGACACCGCCGTGT ACTACTGCGCCGCCGACAACGACCTGCTGTCCGGCCTGGCCGACGAGGACGCCGACTGGGGCCAGGGCAC CCAGGTGACCGTGTCCTCC (369 bp)

[0668] CLL-A12 VHH

[0669] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO: 195

[0670] FR1 EVHLVESGGGLVQPGGSLRLSCSASGRTFS 1 - 30 30 SEQ ID NO: 197 CDR1 QYGVG 31 - 35 5 SEQ ID NO:198 FR2 WSRQAPGKEREIVA 36 - 49 14 SEQ ID NO: 199

[0671]

[0672] CDR2 AIIWSDGSTYYADSVKG 50 - 66 17 SEQ ID N0:200 FR3 RFTISRDNAKNTAYLQMNSLKPEDTAVYYCAA 67 - 98 32 SEQ ID NO:201 CDR3 DNDLLSGLADEDAD 99 - 112 14 SEQ ID NO:202 FR4 WGQGTQVTVSS 113 - 11 SEQ ID 123 NO:203

[0673]

[0674] 123

[0675] CLL-A12 VHH

[0676] Region DNA sequence SEQ ID NO FR1 GAAGTGCACCTGGTGGAGTCCGGAGGAGGATTGGTGCAG SEQ ID CCTGGAGGAAGCCTGAGGCTGAGTTGTAGTGCCAGCGGAA NO:204 GGACATTTTCC CDR1 CAGTACGGCGTGGGG SEQ ID NO:205 FR2 TGGAGCAGGCAGGCTCCTGGAAAAGAGAGGGAGATTGTG SEQ ID GCC NO:206 CDR2 GCCATCATTTGGAGCGACGGGTCTACATACTACGCCGACAG SEQ ID TGTGAAGGGG NO:207 FR3 CGGTTCACCATCAGCAGGGACAATGCCAAAAACACCGCCT SEQ ID ACCTGCAGATGAACTCCCTGAAGCCCGAGGACACCGCCGT NO:208 GTACTACTGCGCCGCC CDR3 GACAACGACCTGCTGTCCGGCCTGGCCGACGAGGACGCC SEQ ID GAC NO:209 FR4 TGGGGCCAGGGCACCCAGGTGACCGTGTCCTCC SEQ ID

[0677] NO:210

[0678]

[0679] (III) Anti-CLL-1 mono-CARs

[0680] CLL-A01 CAR (without signal peptide) - Amino Acids (SEQ ID NO:211):

[0681] AVQLVESGGGLVQVGGSLRLSCVASGRTFSSYITMGWFRQAPGKEREFVASIIGNGGFTYYAASVKGRFT ISRENAKNSLYLQMDDLKPEDTAIYYCAADRGWTRALGARGDEYEDWGQGTQVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (360 aa)

[0682] CLL-A01 CAR (without signal peptide) - Nucleotides (SEQ ID NO:212):

[0683] GCTGTTCAGTTGGTGGAGTCCGGAGGAGGATTGGTGCAGGTGGGAGGATCTTTGAGGTTGAGTTGCGTTG CTTCTGGGAGGACTTTTTCTTCTTATATTACTATGGGATGGTTCAGGCAGGCTCCTGGGAAGGAGAGGGA GTTTGTGGCTTCTATCATTGGAAACGGCGGGTTCACCTACTACGCTGCCAGCGTTAAGGGCAGGTTCACT ATCTCCCGGGAGAACGCCAAGAACAGCTTGTATCTGCAGATGGACGATCTCAAGCCCGAGGACACCGCTA TCTATTACTGCGCCGCCGACAGGGGATGGACCAGAGCTTTGGGTGCCAGGGGTGACGAGTACGAAGACTG GGGACAGGGGACCCAGGTGACAGTTTCCTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACG CCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGT GCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGC GCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGA AAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCT GTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTAC GATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGA GCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGAC GCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (1083 bp)

[0684] CLL-A01 CAR (with signal peptide) - Amino Acids (SEQ ID NO:213):

[0685] MALPVTALLLPLALLLHAARPAVQLVESGGGLVQVGGSLRLSCVASGRTFSSYITMGWFRQAPGKEREFV ASIIGNGGFTYYAASVKGRFTISRENAKNSLYLQMDDLKPEDTAIYYCAADRGWTRALGARGDEYEDWGQ GTQVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL AGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR (381 aa)

[0686] CLL-A01 CAR (with signal peptide) - Nucleotides (SEQ ID NO:214)ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCTGTTC AGTTGGTGGAGTCCGGAGGAGGATTGGTGCAGGTGGGAGGATCTTTGAGGTTGAGTTGCGTTGCTTCTGG GAGGACTTTTTCTTCTTATATTACTATGGGATGGTTCAGGCAGGCTCCTGGGAAGGAGAGGGAGTTTGTG GCTTCTATCATTGGAAACGGCGGGTTCACCTACTACGCTGCCAGCGTTAAGGGCAGGTTCACTATCTCCC GGGAGAACGCCAAGAACAGCTTGTATCTGCAGATGGACGATCTCAAGCCCGAGGACACCGCTATCTATTA CTGCGCCGCCGACAGGGGATGGACCAGAGCTTTGGGTGCCAGGGGTGACGAGTACGAAGACTGGGGACAG GGGACCCAGGTGACAGTTTCCTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGC CGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCC AGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTG GCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAAC TCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCC GCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCT GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTC ACATGCAGGCCCTGCCCCCTCGCTAA (1146 bp)

[0687] CLL-A02 CAR (without signal peptide) - Amino Acids (SEQ ID NO:215):

[0688] QLQLVESGGGLVQAGGSLRLSCAASGRTFSNYIMGWFRQAPGKEREFVAAITWSGGSTYYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCAARDDPLLLLRLWMLWNEYDYWGQGTQVTVSSFVPVFLPAKPTT TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRG RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR (361 aa)

[0689] CLL-A02 CAR (without signal peptide) - Nucleotides (SEQ ID NO:216):

[0690] CAGTTGCAGCTCGTGGAGTCCGGAGGAGGACTCGTTCAGGCAGGAGGATCACTCAGGCTCTCCTGCGCTG CTTCCGGAAGAACCTTCTCCAACTACATCATGGGCTGGTTCAGGCAGGCCCCCGGAAAAGAAAGGGAGTT CGTGGCTGCCATCACCTGGAGCGGAGGTTCTACCTACTACGCCGACTCCGTCAAGGGCAGATTCACAATC AGCAGGGACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCTTGAAGCCCGAGGACACCGCCGTGT ACTACTGCGCTGCTAGGGATGACCCCCTGCTGTTGCTGCGTCTGTGGATGCTGTGGAACGAGTACGACTA CTGGGGCCAGGGCACCCAGGTTACTGTGTCTAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGG CGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTG GGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGC AGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATG GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGC AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAG TACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTC AGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGG CGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC GACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (1086 bp)

[0691] CLL-A02 CAR (with signal peptide) - Amino Acids (SEQ ID NO:217):

[0692] MALPVTALLLPLALLLHAARPQLQLVESGGGLVQAGGSLRLSCAASGRTFSNYIMGWFRQAPGKEREFVA AITWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARDDPLLLLRLWMLWNEYDYWG QGTQVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR (382 aa)

[0693] CLL-A02 CAR (with signal peptide) - Nucleotides (SEQ ID NO:218):

[0694] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGTTGC AGCTCGTGGAGTCCGGAGGAGGACTCGTTCAGGCAGGAGGATCACTCAGGCTCTCCTGCGCTGCTTCCGG AAGAACCTTCTCCAACTACATCATGGGCTGGTTCAGGCAGGCCCCCGGAAAAGAAAGGGAGTTCGTGGCT GCCATCACCTGGAGCGGAGGTTCTACCTACTACGCCGACTCCGTCAAGGGCAGATTCACAATCAGCAGGG ACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCTTGAAGCCCGAGGACACCGCCGTGTACTACTG CGCTGCTAGGGATGACCCCCTGCTGTTGCTGCGTCTGTGGATGCTGTGGAACGAGTACGACTACTGGGGC CAGGGCACCCAGGTTACTGTGTCTAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAG CGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCG GCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCC TTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGA AACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAG CTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCC CCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGC CGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGCTAA (1149 bp)

[0695] CLL-A03 CAR (without signal peptide) - Amino Acids (SEQ ID NO:219):

[0696] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEHEFVAAISWSGGSTYYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCAARDGGFYDSDWRRSRNEYDYWGQGTQVTVSSFVPVFLPAKPTT TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRG RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR (361 aa)

[0697] CLL-A03 CAR (without signal peptide) - Nucleotides (SEQ ID NO:220):

[0698] GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTGCAGGCAGGAGGAAGCCTGAGGCTGAGTTGTGCCG CTAGCGGAAGGACATTTAGTAGCTACGCCATGGGATGGTTTAGGCAGGCCCCAGGAAAGGAGCACGAGTT CGTGGCTGCAATTTCCTGGAGCGGAGGAAGCACATATTACGCCGATTCTGTGAAGGGCAGGTTCACCATC TCCCGGGACAACGCTAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCCGAAGACACCGCCGTGT ACTACTGCGCTGCCAGAGACGGAGGATTCTACGACTCCGACTGGAGGAGGAGCAGAAACGAGTACGACTA CTGGGGCCAGGGAACCCAGGTGACAGTGTCTAGCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACG ACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGG CGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTG GGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGC AGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATG GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGC AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAG TACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTC AGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGG CGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC GACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (1086 bp)

[0699] CLL-A03 CAR (with signal peptide) - Amino Acids (SEQ ID NO:221):

[0700] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEHEFVAAISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARDGGFYDSDWRRSRNEYDYWG QGTQVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR (382 aa)

[0701] CLL-A03 CAR (with signal peptide) - Nucleotides (SEQ ID NO:222):

[0702] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAAGTGC AGCTGGTGGAGAGCGGAGGAGGATTGGTGCAGGCAGGAGGAAGCCTGAGGCTGAGTTGTGCCGCTAGCGG AAGGACATTTAGTAGCTACGCCATGGGATGGTTTAGGCAGGCCCCAGGAAAGGAGCACGAGTTCGTGGCT GCAATTTCCTGGAGCGGAGGAAGCACATATTACGCCGATTCTGTGAAGGGCAGGTTCACCATCTCCCGGG ACAACGCTAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCCGAAGACACCGCCGTGTACTACTG CGCTGCCAGAGACGGAGGATTCTACGACTCCGACTGGAGGAGGAGCAGAAACGAGTACGACTACTGGGGC CAGGGAACCCAGGTGACAGTGTCTAGCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAG CGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCG GCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCC TTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGA AACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAG CTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCC CCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGC CGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGCTAA(1149 bp)

[0703] CLL-A04 CAR (without signal peptide) - Amino Acids (SEQ ID NO:223):

[0704] EVQLVESGGGLVQVGGSLRLSCVASGRTFSSYITMGWFRQAPGKEREFVASIIGNDAFTYYAASVKGRFT ISRENAKNSLYLQMDDLKPEDTATYFCAADRGWTAALGARGDHYEDWGQGTQVTVSSFVPVFLPAKPTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (360 aa)

[0705] CLL-A04 CAR (without signal peptide) - Nucleotides (SEQ ID NO:224)GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTGCAGGTGGGAGGAAGCCTGAGGCTGAGTTGTGTGG CTAGCGGGAGGACATTTAGTTCCTATATCACAATGGGGTGGTTCAGGCAGGCTCCAGGAAAGGAGAGGGA GTTCGTGGCTAGTATTATTGGAAACGACGCCTTCACCTACTACGCTGCCTCAGTGAAGGGCCGGTTCACC ATCAGCAGGGAGAATGCTAAGAACAGCCTGTACCTGCAGATGGATGATTTGAAGCCCGAGGACACAGCTA CCTACTTCTGCGCCGCCGACCGAGGATGGACAGCTGCTTTGGGAGCACGGGGAGACCACTACGAGGACTG GGGACAGGGAACACAGGTGACCGTGAGCTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACG CCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGT GCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGC GCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGA AAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCT GTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTAC GATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGA GCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGAC GCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (1083 bp)

[0706] CLL-A04 CAR (with signal peptide) - Amino Acids (SEQ ID NO:225):

[0707] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQVGGSLRLSCVASGRTFSSYITMGWFRQAPGKEREFV ASIIGNDAFTYYAASVKGRFTISRENAKNSLYLQMDDLKPEDTATYFCAADRGWTAALGARGDHYEDWGQ GTQVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL AGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR (381 aa)

[0708] CLL-A04 CAR (with signal peptide) - Nucleotides (SEQ ID NO:226):

[0709] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAAGTGC AGCTGGTGGAGAGCGGAGGAGGATTGGTGCAGGTGGGAGGAAGCCTGAGGCTGAGTTGTGTGGCTAGCGG GAGGACATTTAGTTCCTATATCACAATGGGGTGGTTCAGGCAGGCTCCAGGAAAGGAGAGGGAGTTCGTG GCTAGTATTATTGGAAACGACGCCTTCACCTACTACGCTGCCTCAGTGAAGGGCCGGTTCACCATCAGCA GGGAGAATGCTAAGAACAGCCTGTACCTGCAGATGGATGATTTGAAGCCCGAGGACACAGCTACCTACTT CTGCGCCGCCGACCGAGGATGGACAGCTGCTTTGGGAGCACGGGGAGACCACTACGAGGACTGGGGACAG GGAACACAGGTGACCGTGAGCTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCC AGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTG GCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAAC TCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCC GCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCT GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTC ACATGCAGGCCCTGCCCCCTCGCTAA (1146 bp)

[0710] CLL-A05 CAR (without signal peptide) - Amino Acids (SEQ ID NO:227):

[0711] QLHFVESGGGLVQAGGSLRLSCAASGRTFSSYITMGWFRQAPGKEREFVASIIGNGGFTYYAASVKGRFT ISRDNAKNSLYLQMDSLKPEDTAVYYCAADRGWTADLGTRGDDYEDWGQGTQVTVSSFVPVFLPAKPTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (360 aa)

[0712] CLL-A05 CAR (without signal peptide) - Nucleotides (SEQ ID NO:228):

[0713] CAGTTGCACTTCGTGGAGTCCGGCGGAGGACTCGTTCAGGCTGGAGGATCACTCAGGCTCTCCTGCGCTG CTTCCGGAAGAACCTTCTCCTCCTACATCACCATGGGCTGGTTCAGGCAGGCCCCTGGAAAGGAACGCGA GTTTGTGGCCAGTATCATTGGAAACGGGGGCTTCACTTACTACGCCGCCTCCGTTAAGGGCAGGTTCACC ATCTCCCGGGACAACGCTAAGAACAGCCTGTACTTGCAGATGGACAGCCTGAAGCCTGAGGACACCGCTG TGTACTACTGTGCAGCCGACAGAGGATGGACCGCAGATCTGGGTACAAGGGGCGACGACTACGAAGACTG GGGACAGGGAACCCAGGTGACCGTTTCCTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACG CCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGT GCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGC GCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGA AAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCT GTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGA GCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGAC GCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (1083 bp)

[0714] CLL-A05 CAR (with signal peptide) - Amino Acids (SEQ ID NO:229):

[0715] MALPVTALLLPLALLLHAARPQLHFVESGGGLVQAGGSLRLSCAASGRTFSSYITMGWFRQAPGKEREFV ASIIGNGGFTYYAASVKGRFTISRDNAKNSLYLQMDSLKPEDTAVYYCAADRGWTADLGTRGDDYEDWGQ GTQVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL AGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR (381 aa)

[0716] CLL-A05 CAR (with signal peptide) - Nucleotides (SEQ ID NO:230):

[0717] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGTTGC ACTTCGTGGAGTCCGGCGGAGGACTCGTTCAGGCTGGAGGATCACTCAGGCTCTCCTGCGCTGCTTCCGG AAGAACCTTCTCCTCCTACATCACCATGGGCTGGTTCAGGCAGGCCCCTGGAAAGGAACGCGAGTTTGTG GCCAGTATCATTGGAAACGGGGGCTTCACTTACTACGCCGCCTCCGTTAAGGGCAGGTTCACCATCTCCC GGGACAACGCTAAGAACAGCCTGTACTTGCAGATGGACAGCCTGAAGCCTGAGGACACCGCTGTGTACTA CTGTGCAGCCGACAGAGGATGGACCGCAGATCTGGGTACAAGGGGCGACGACTACGAAGACTGGGGACAG GGAACCCAGGTGACCGTTTCCTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGC CGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCC AGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTG GCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAAC TCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCC GCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCT GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTC ACATGCAGGCCCTGCCCCCTCGCTAA (1146 bp)

[0718] CLL-A06 CAR (without signal peptide) - Amino Acids (SEQ ID NO:231):

[0719] ELQLVESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTIS RDNAKNTVYLQMNSLKVEDTAVYYCNEVPPYGTSWYAEDEYWGQGTQVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR (355 aa)

[0720] CLL-A06 CAR (without signal peptide) - Nucleotides (SEQ ID NO:232):

[0721] GAACTGCAGCTGGTGGAATCCGGCGGAGGATTGGTGCAGCCTGGAGGATCTTTGAGGCTGAGCTGCGCCG CTTCCGGAAGTATCTTTAGTATTAACGCCATGGGATGGTACAGGCAGGCCCCTGGAAAGCAGAGGGAGTT GGTGGCTGCTATTACCTCCGGAGGAAGCACCAACTACGCAGACAGCGTGAAGGGGAGGTTCACCATCAGC AGGGATAACGCAAAGAACACCGTCTACCTGCAGATGAACAGCCTGAAGGTGGAAGATACCGCAGTGTACT ACTGCAACGAAGTGCCCCCCTACGGCACAAGTTGGTACGCAGAGGACGAGTACTGGGGACAGGGCACACA GGTGACCGTGAGTTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCA CCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGG GGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGAC TTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTAT ATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTC CAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCA GCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG AGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATG AACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGCTAA (1068 bp)

[0722] CLL-A06 CAR (with signal peptide) - Amino Acids (SEQ ID NO:233):

[0723] MALPVTALLLPLALLLHAARPELQLVESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVA AITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKVEDTAVYYCNEVPPYGTSWYAEDEYWGQGTQVT VSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (376 aa)

[0724] CLL-A06 CAR (with signal peptide) - Nucleotides (SEQ ID NO:234)ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAACTGC AGCTGGTGGAATCCGGCGGAGGATTGGTGCAGCCTGGAGGATCTTTGAGGCTGAGCTGCGCCGCTTCCGG AAGTATCTTTAGTATTAACGCCATGGGATGGTACAGGCAGGCCCCTGGAAAGCAGAGGGAGTTGGTGGCT GCTATTACCTCCGGAGGAAGCACCAACTACGCAGACAGCGTGAAGGGGAGGTTCACCATCAGCAGGGATA ACGCAAAGAACACCGTCTACCTGCAGATGAACAGCCTGAAGGTGGAAGATACCGCAGTGTACTACTGCAA CGAAGTGCCCCCCTACGGCACAAGTTGGTACGCAGAGGACGAGTACTGGGGACAGGGCACACAGGTGACC GTGAGTTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACAC CGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCG C AGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGG GTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCA AACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGA AGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGC CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGC CCCCTCGCTAA (1131 bp)

[0725] CLL-A07 CAR (without signal peptide) - Amino Acids (SEQ ID NO:235):

[0726] AVQLVESGGGLVQPGGSLRLFCAASGFTFGSYDMSWVRQAPGKEVEWVSTINSRGDRTYYPDSVKGRFTI SRDNAKNTVSLQMNNLKPEDTAVYYCATQETPGGDEAYWGQGTQVTVSSFVPVFLPAKPTTTPAPRPPTP APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFK QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (352 aa)

[0727] CLL-A07 CAR (without signal peptide) - Nucleotides (SEQ ID NO:236):

[0728] GCTGTTCAGTTGGTGGAGTCCGGAGGAGGATTGGTGCAGCCTGGTGGATCTTTGAGGTTGTTTTGTGCTG CTTCTGGGTTTACCTTTGGATCTTATGATATGTCCTGGGTGAGGCAGGCCCCCGGAAAAGAAGTGGAGTG GGTGAGCACTATTAACTCCAGGGGGGACAGGACATACTACCCCGATTCCGTTAAGGGCCGGTTTACAATC TCCCGTGACAACGCTAAGAACACCGTGAGCCTGCAGATGAATAACTTGAAGCCCGAGGACACCGCCGTGT ACTACTGTGCTACCCAGGAAACCCCCGGCGGAGATGAAGCCTATTGGGGACAGGGAACACAGGTGACCGTGAGTTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCG GCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCA G TGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGT CCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAA C AAC C ATTTATG AG AC C AGTAC AAACTACTC AAG AG GAAG ATG GCTGTAG CTGC C G ATTTC C AG AAGAAG AAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGC CGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGA AAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGA TGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCC CCTCGCTAA (1059 bp)

[0729] CLL-A07 CAR (with signal peptide) - Amino Acids (SEQ ID NO:237):

[0730] MALPVTALLLPLALLLHAARPAVQLVESGGGLVQPGGSLRLFCAASGFTFGSYDMSWVRQAPGKEVEWVS TINSRGDRTYYPDSVKGRFTISRDNAKNTVSLQMNNLKPEDTAVYYCATQETPGGDEAYWGQGTQVTVSS FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR (373 aa)

[0731] CLL-A07 CAR (with signal peptide) - Nucleotides (SEQ ID NO:238):

[0732] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCTGTTC AGTTGGTGGAGTCCGGAGGAGGATTGGTGCAGCCTGGTGGATCTTTGAGGTTGTTTTGTGCTGCTTCTGG GTTTACCTTTGGATCTTATGATATGTCCTGGGTGAGGCAGGCCCCCGGAAAAGAAGTGGAGTGGGTGAGC ACTATTAACTCCAGGGGGGACAGGACATACTACCCCGATTCCGTTAAGGGCCGGTTTACAATCTCCCGTG ACAACGCTAAGAACACCGTGAGCCTGCAGATGAATAACTTGAAGCCCGAGGACACCGCCGTGTACTACTG TGCTACCCAGGAAACCCCCGGCGGAGATGAAGCCTATTGGGGACAGGGAACACAGGTGACCGTGAGTTCC TTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCA CCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACAC GAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTC CTGTC ACTGGTTATC AC C CTTTACTGC AAAC GG GG C AG AAAGAAACTC CTGTATATATTC AAAC AAC CAT TTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGG AGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACC CTGAGATGG GG GG AAAG CC GAG AAG G AAG AAC C CTC AG GAAG GC CTGTAC AATGAACTG C AG AAAG ATAA GATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTT TACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCT AA (1122 bp)

[0733] CLL-A08 CAR (without signal peptide) - Amino Acids (SEQ ID NO:239):

[0734] EVHLVESGEGLVQVGGSLRLSCVASGRTFSSYITMGWFRQAPGKEREFVASIIGNGGFTYYAASVKGRFT ISRENAKNSLYLQMDDLKPEDTAIYYCAADRGWTAALGARGDDYEDWGQGTQVTVSSFVPVFLPAKPTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (360 aa)

[0735] CLL-A08 CAR (without signal peptide) - Nucleotides (SEQ ID NO:240):

[0736] GAAGTGCACCTGGTGGAGTCCGGAGAGGGATTGGTGCAGGTGGGAGGAAGCCTGAGGCTGAGTTGTGTGG CCAGTGGCAGGACATTTAGCAGCTACATCACAATGGGGTGGTTTAGGCAGGCCCCCGGAAAGGAAAGGGA GTTTGTGGCTAGTATTATCGGAAATGGCGGCTTCACTTACTACGCCGCATCCGTTAAGGGCAGGTTCACC ATCAGCAGGGAGAACGCCAAGAACAGCCTGTACTTGCAGATGGACGACCTGAAGCCTGAGGACACCGCTA TCTATTACTGCGCAGCCGACAGGGGATGGACCGCTGCTTTGGGAGCAAGGGGAGACGACTACGAAGACTG GGGACAGGGCACCCAGGTGACAGTTTCCTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACG CCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGT GCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGC GCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGA AAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCT GTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTAC GATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGA GCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGAC GCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (1083 bp)CLL-A08 CAR (with signal peptide) - Amino Acids (SEQ ID NO:241):

[0737] MALPVTALLLPLALLLHAARPEVHLVESGEGLVQVGGSLRLSCVASGRTFSSYITMGWFRQAPGKEREFV ASIIGNGGFTYYAASVKGRFTISRENAKNSLYLQMDDLKPEDTAIYYCAADRGWTAALGARGDDYEDWGQ GTQVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL AGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR (381 aa)

[0738] CLL-A08 CAR (with signal peptide) - Nucleotides (SEQ ID NO:242):

[0739] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAAGTGC ACCTGGTGGAGTCCGGAGAGGGATTGGTGCAGGTGGGAGGAAGCCTGAGGCTGAGTTGTGTGGCCAGTGG CAGGACATTTAGCAGCTACATCACAATGGGGTGGTTTAGGCAGGCCCCCGGAAAGGAAAGGGAGTTTGTG GCTAGTATTATCGGAAATGGCGGCTTCACTTACTACGCCGCATCCGTTAAGGGCAGGTTCACCATCAGCA GGGAGAACGCCAAGAACAGCCTGTACTTGCAGATGGACGACCTGAAGCCTGAGGACACCGCTATCTATTA CTGCGCAGCCGACAGGGGATGGACCGCTGCTTTGGGAGCAAGGGGAGACGACTACGAAGACTGGGGACAG GGCACCCAGGTGACAGTTTCCTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGC CGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCC AGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTG GCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAAC TCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCC GCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCT GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTC ACATGCAGGCCCTGCCCCCTCGCTAA (1146 bp)

[0740] CLL-A09 CAR (without signal peptide) - Amino Acids (SEQ ID NO:243):

[0741] QVQLVESGGGLVQPGGSLRLSCAASGRTFSMDAMGWFRQAPGKEREFVALISWKSDSTFYADSVKGRFTI SRDNAKNTVSLQMNSLKPEDTAVYYCAVGYIATMSQGTMDYWGQGTQVTVSSFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR (355 aa)CLL-A09 CAR (without signal peptide) - Nucleotides (SEQ ID NO:244):

[0742] CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGCCAGGAGGATCACTCAGGCTGTCATGCGCCG CTTCCGGAAGAACATTCTCCATGGACGCAATGGGCTGGTTCAGGCAGGCTCCTGGAAAGGAGAGGGAGTT CGTGGCACTGATTTCCTGGAAGTCCGACTCCACATTCTACGCTGATAGTGTGAAAGGACGGTTCACCATC TCCCGGGACAATGCTAAGAACACCGTGAGCCTGCAGATGAACAGCCTGAAGCCCGAAGACACCGCCGTGT ACTACTGCGCAGTTGGCTACATTGCCACCATGAGCCAGGGCACCATGGACTACTGGGGACAGGGAACCCA GGTGACCGTGTCTTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCA CCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGG GGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGAC TTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTAT ATATTC AAAC AAC C ATTTATGAG AC C AGTAC AAACTACTCAAG AG GAAG ATGG CTGTAG CTGC C G ATTTC CAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCA GCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG AGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATG AACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGCTAA (1068 bp)

[0743] CLL-A09 CAR (with signal peptide) - Amino Acids (SEQ ID NO:245):

[0744] MALPVTALLLPLALLLHAARPQVQLVESGGGLVQPGGSLRLSCAASGRTFSMDAMGWFRQAPGKEREFVA LISWKSDSTFYADSVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCAVGYIATMSQGTMDYWGQGTQVT VSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (376 aa)

[0745] CLL-A09 CAR (with signal peptide) - Nucleotides (SEQ ID NO:246):

[0746] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTTC AGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGCCAGGAGGATCACTCAGGCTGTCATGCGCCGCTTCCGG AAGAACATTCTCCATGGACGCAATGGGCTGGTTCAGGCAGGCTCCTGGAAAGGAGAGGGAGTTCGTGGCA CTGATTTCCTGGAAGTCCGACTCCACATTCTACGCTGATAGTGTGAAAGGACGGTTCACCATCTCCCGGG ACAATGCTAAGAACACCGTGAGCCTGCAGATGAACAGCCTGAAGCCCGAAGACACCGCCGTGTACTACTGCGCAGTTGGCTACATTGCCACCATGAGCCAGGGCACCATGGACTACTGGGGACAGGGAACCCAGGTGACC GTGTCTTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACAC CGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCG C AGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGG GTC CTTCTC CTGTC ACTGGTTATC AC C CTTTACTG C AAAC GG GG C AG AAAG AAACTC CTGTATATATTC A AAC AACC ATTTATG AG AC C AGTAC AAACTACTC AAG AG GAAG ATG GCTGTAGCTG C C GATTTC C AGAAG A AGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGC C AGAAC C AGCTCTATAAC GAG CTC AATCTAG GAC GAAGAGAGG AGTAC G ATGTTTTGGAC AAGAGAC GTG GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGC CCCCTCGCTAA (1131 bp)

[0747] CLL-A10 CAR (without signal peptide) - Amino Acids (SEQ ID NO:247):

[0748] AVQLVESGGGLVQPGDSLRLFCAASGFTFGTYDMSWVRQAPGKEVEWVSTINSRGDRTYYPDSVKGRFTI SRDNAKNTLYLQMDSLKPEDTALYYCVTDAFGSWGQGTQVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (347 aa)

[0749] CLL-A10 CAR (without signal peptide) - Nucleotides (SEQ ID NO:248):

[0750] GCTGTTCAGTTGGTGGAGTCCGGAGGAGGATTGGTGCAGCCTGGTGATTCCCTGAGGTTGTTTTGTGCTG CTTCTGGGTTTACCTTTGGAACTTATGATATGTCCTGGGTGAGGCAGGCCCCTGGAAAGGAAGTTGAGTG GGTGAGTACCATTAATTCCAGGGGGGACAGGACATACTACCCTGATTCCGTCAAGGGCCGCTTTACAATC TCCCGCGACAACGCTAAGAACACCCTGTACCTGCAGATGGATAGTCTGAAACCCGAGGACACAGCCTTGT ATTATTGCGTGACCGACGCCTTCGGCTCCTGGGGACAGGGAACACAGGTGACAGTGTCCTCCTTCGTGCC GGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCG TCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGG C TGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACT GGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGA CCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGG AGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGG TCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (1044 bp)

[0751] CLL-A10 CAR (with signal peptide) - Amino Acids (SEQ ID NO:249):

[0752] MALPVTALLLPLALLLHAARPAVQLVESGGGLVQPGDSLRLFCAASGFTFGTYDMSWVRQAPGKEVEWVS TINSRGDRTYYPDSVKGRFTISRDNAKNTLYLQMDSLKPEDTALYYCVTDAFGSWGQGTQVTVSSFVPVF LPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVI TLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR (368 aa)

[0753] CLL-A10 CAR (with signal peptide) - Nucleotides (SEQ ID NO:250):

[0754] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCTGTTC AGTTGGTGGAGTCCGGAGGAGGATTGGTGCAGCCTGGTGATTCCCTGAGGTTGTTTTGTGCTGCTTCTGG GTTTACCTTTGGAACTTATGATATGTCCTGGGTGAGGCAGGCCCCTGGAAAGGAAGTTGAGTGGGTGAGT ACCATTAATTCCAGGGGGGACAGGACATACTACCCTGATTCCGTCAAGGGCCGCTTTACAATCTCCCGCG ACAACGCTAAGAACACCCTGTACCTGCAGATGGATAGTCTGAAACCCGAGGACACAGCCTTGTATTATTG CGTGACCGACGCCTTCGGCTCCTGGGGACAGGGAACACAGGTGACAGTGTCCTCCTTCGTGCCGGTCTTC CTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGC CCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTT CGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATC ACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTAC AAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAG AGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTA CAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGT ACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (1107 bp)

[0755] CLL-A11 CAR (without signal peptide) - Amino Acids (SEQ ID NO:251):

[0756] EVDLVESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGNQRELVAAITSGANTAYSDSVKGRFTIS RDNAKNTVYLQMNSLKPDDTAVYYCYALIPLNGSSWPPDEYWGQGTQVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR (355 aa)

[0757] CLL-A11 CAR (without signal peptide) - Nucleotides (SEQ ID NO:252):

[0758] GAAGTGGACCTGGTGGAGAGCGGAGGAGGATTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGTTGTGCCG CAAGCGGAAGCATCTTTAGCATCAACGCTATGGGGTGGTACAGGCAGGCACCCGGAAACCAGAGGGAGTT GGTGGCTGCTATTACCTCTGGAGCTAACACCGCATACTCCGACAGCGTGAAGGGAAGGTTCACTATCTCC AGAGATAACGCTAAGAACACCGTCTACCTCCAGATGAACAGCCTGAAGCCTGATGATACCGCCGTTTACT ACTGCTACGCCCTGATTCCCTTGAATGGCTCCTCCTGGCCCCCCGATGAATACTGGGGACAGGGAACCCA GGTGACCGTCTCTTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCA CCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGG GGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGAC TTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTAT ATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTC CAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCA GCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG AGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATG AACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGCTAA (1068 bp)

[0759] CLL-A11 CAR (with signal peptide) - Amino Acids (SEQ ID NO:253):

[0760] MALPVTALLLPLALLLHAARPEVDLVESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGNQRELVA AITSGANTAYSDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCYALIPLNGSSWPPDEYWGQGTQVT VSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (376 aa)

[0761] CLL-A11 CAR (with signal peptide) - Nucleotides (SEQ ID NO:254)ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAAGTGG ACCTGGTGGAGAGCGGAGGAGGATTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGTTGTGCCGCAAGCGG AAGCATCTTTAGCATCAACGCTATGGGGTGGTACAGGCAGGCACCCGGAAACCAGAGGGAGTTGGTGGCT GCTATTACCTCTGGAGCTAACACCGCATACTCCGACAGCGTGAAGGGAAGGTTCACTATCTCCAGAGATA AC GCTAAG AAC AC C GTCTAC CTC C AG ATG AAC AG CCTG AAG CCTGATGATAC C GC CGTTTACTACTG CTA CGCCCTGATTCCCTTGAATGGCTCCTCCTGGCCCCCCGATGAATACTGGGGACAGGGAACCCAGGTGACC GTCTCTTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACAC CGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCG C AGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGG GTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCA AACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGA AGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGC CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGC CCCCTCGCTAA (1131 bp)

[0762] CLL-A12 CAR (without signal peptide) - Amino Acids (SEQ ID NO:255):

[0763] EVHLVESGGGLVQPGGSLRLSCSASGRTFSQYGVGWSRQAPGKEREIVAAIIWSDGSTYYADSVKGRFTI SRDNAKNTAYLQMNSLKPEDTAVYYCAADNDLLSGLADEDADWGQGTQVTVSSFVPVFLPAKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR (356 aa)

[0764] CLL-A12 CAR (without signal peptide) - Nucleotides (SEQ ID NO:256):

[0765] GAAGTGCACCTGGTGGAGTCCGGAGGAGGATTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGTTGTAGTG CCAGCGGAAGGACATTTTCCCAGTACGGCGTGGGGTGGAGCAGGCAGGCTCCTGGAAAAGAGAGGGAGAT TGTGGCCGCCATCATTTGGAGCGACGGGTCTACATACTACGCCGACAGTGTGAAGGGGCGGTTCACCATC AGCAGGGACAATGCCAAAAACACCGCCTACCTGCAGATGAACTCCCTGAAGCCCGAGGACACCGCCGTGT ACTACTGCGCCGCCGACAACGACCTGCTGTCCGGCCTGGCCGACGAGGACGCCGACTGGGGCCAGGGCACCCAGGTGACCGTGTCCTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGA CCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGG CGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGG GACTTGTG GG GTCCTTCTCCTGTC ACTG GTTATC AC C CTTTACTG C AAAC G GG GC AG AAAG AAACTC CTG TATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGAT TTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTA CCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACA ATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG CAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATG CAGGCCCTGCCCCCTCGCTAA (1071 bp)

[0766] CLL-A12 CAR (with signal peptide) - Amino Acids (SEQ ID NO:257):

[0767] MALPVTALLLPLALLLHAARPEVHLVESGGGLVQPGGSLRLSCSASGRTFSQYGVGWSRQAPGKEREIVA AIIWSDGSTYYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCAADNDLLSGLADEDADWGQGTQV TVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (377 aa)

[0768] CLL-A12 CAR (with signal peptide) - Nucleotides (SEQ ID NO:258):

[0769] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAAGTGC ACCTGGTGGAGTCCGGAGGAGGATTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGTTGTAGTGCCAGCGG AAGGACATTTTCCCAGTACGGCGTGGGGTGGAGCAGGCAGGCTCCTGGAAAAGAGAGGGAGATTGTGGCC GCCATCATTTGGAGCGACGGGTCTACATACTACGCCGACAGTGTGAAGGGGCGGTTCACCATCAGCAGGG ACAATGCCAAAAACACCGCCTACCTGCAGATGAACTCCCTGAAGCCCGAGGACACCGCCGTGTACTACTG CGCCGCCGACAACGACCTGCTGTCCGGCCTGGCCGACGAGGACGCCGACTGGGGCCAGGGCACCCAGGTG ACCGTGTCCTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAA CACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGG G CGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGT GGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATAT TCAAAC AACC ATTTATGAGAC C AGTAC AAACTACTC AAGAG GAAG ATG GCTGTAGCTGC C GATTTC GAGA AGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGAC GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACT GCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGG CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCC TGCCCCCTCGCTAA (1134 bp)

[0770] (IV) Anti-CD33 VHHs

[0771] CD33-B01 VHH - Amino Acids (SEQ ID NO:259):

[0772] EVQLVESGGGLVQAGGSLRLTCVASGSLLRFGAIGWHRQGPEKQRALVASITDGHTNYADSVKGRFTISR DNAKNTVYLQMNNLKTEDTGIYYCSGATLSLNLRWKYDYWGQGIQVTVSS (120 aa)

[0773] CD33-B01 VHH - Nucleotides (SEQ ID NO:260):

[0774] GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTGCAGGCAGGAGGAAGCCTGAGGCTGACATGTGTGG CCAGCGGAAGCCTGCTGAGGTTTGGAGCAATCGGATGGCACAGGCAGGGACCTGAGAAGCAGAGGGCACT GGTGGCTAGCATCACCGATGGCCATACCAATTACGCCGACTCCGTGAAAGGAAGGTTCACCATCTCCAGG GACAACGCCAAGAACACCGTGTACTTGCAGATGAACAACCTGAAGACCGAGGACACAGGCATCTACTACT GCTCCGGCGCCACCTTGAGCCTGAACTTGCGATGGAAGTACGACTACTGGGGCCAGGGAATCCAGGTGAC CGTGTCTTCC (360 bp)

[0775] CD33-B01 VHH

[0776] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO: ID NO:259

[0777] FR1 EVQLVESGGGLVQAGGSLRLTCVASGSLLR 1 - 30 30 SEQ ID NO:261 CDR1 FGAIG 31 - 35 5 SEQ ID NO:262 FR2 WHRQGPEKQRALVA 36 - 49 14 SEQ ID NO:263 CDR2 SITDGHTNYADSVKG 50 - 64 15 SEQ ID NO:264 FR3 RFTISRDNAKNTVYLQMNNLKTEDTGIYYCSG 65 - 96 32 SEQ ID

[0778] NO:265

[0779]

[0780] CDR3 ATLSLNLRWKYDY 97 - 109 13 SEQ ID NO:266 FR4 WGQGIQVTVSS 110 - 11 SEQ ID 120 NO:267

[0781]

[0782] 120

[0783] CD33-B01 VHH

[0784] Region DNA sequence SEQ ID NO FR1 GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTGCAGG SEQ ID CAGGAGGAAGCCTGAGGCTGACATGTGTGGCCAGCGGAAG NO:268 CCTGCTGAGG CDR1 TTTGGAGCAATCGGA SEQ ID NO:269 FR2 TGGCACAGGCAGGGACCTGAGAAGCAGAGGGCACTGGTGG SEQ ID CT NO:270 CDR2 AGCATCACCGATGGCCATACCAATTACGCCGACTCCGTGAAA SEQ ID GGA NO:271 FR3 AGGTTCACCATCTCCAGGGACAACGCCAAGAACACCGTGTA SEQ ID CTTGCAGATGAACAACCTGAAGACCGAGGACACAGGCATCT NO:272 ACTACTGCTCCGGC CDR3 GCCACCTTGAGCCTGAACTTGCGATGGAAGTACGACTAC SEQ ID NO:273 FR4 TGGGGCCAGGGAATCCAGGTGACCGTGTCTTCC SEQ ID

[0785] NO:274

[0786]

[0787] CD33-B02 VHH - Amino Acids (SEQ ID NO:275):

[0788] EVDLVESGGGLVQPGGSLRLSCAASGFTLDHYAIKWFRQAPGKGREGVSCISSSGGRTNYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCAAAVAVCGGSWSWYNQWGQGTQVTVSS (123 aa)

[0789] CD33-B02 VHH - Nucleotides (SEQ ID NO:276):

[0790] GAAGTGGACCTGGTGGAGAGCGGAGGAGGATTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGTTGTGCCG CAAGCGGATTTACACTGGACCACTACGCCATCAAGTGGTTCAGGCAGGCCCCCGGAAAAGGCAGAGAGGG AGTTAGCTGCATTAGCTCCAGTGGAGGAAGGACCAACTACGCCGATAGTGTGAAGGGCAGATTCACCATC TCCAGGGACAACGCCAAGAACACAGTGTATCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTTTATTACTGCGCAGCCGCCGTGGCTGTGTGCGGAGGATCTTGGTCTTGGTACAACCAGTGGGGGCAGGGCAC CCAGGTTACAGTTTCCTCC (369 bp)

[0791] CD33-B02 VHH

[0792] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO:275

[0793] FR1 EVDLVESGGGLVQPGGSLRLSCAASGFTLD 1 - 30 30 SEQ ID NO:277 CDR1 HYAIK 31 - 35 5 SEQ ID NO:278 FR2 WFRQAPGKGREGVS 36 - 49 14 SEQ ID NO:279 CDR2 CISSSGGRTNYADSVKG 50 - 66 17 SEQ ID NO:280 FR3 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA 67 - 98 32 SEQ ID NO:281 CDR3 AVAVCGGSWSWYNQ 99 - 112 14 SEQ ID NO:282 FR4 WGQGTQVTVSS 113 - 11 SEQ ID 123 NO:283

[0794]

[0795] 123

[0796] CD33-B02 VHH

[0797] Region DNA sequence SEQ ID NO FR1 GAAGTGGACCTGGTGGAGAGCGGAGGAGGATTGGTGCAGCC SEQ ID TGGAGGAAGCCTGAGGCTGAGTTGTGCCGCAAGCGGATTTAC NO:284 ACTGGAC CDR1 CACTACGCCATCAAG SEQ ID NO:285 FR2 TGGTTCAGGCAGGCCCCCGGAAAAGGCAGAGAGGGAGTTAG SEQ ID C NO:286

[0798]

[0799] CDR2 TGCATTAGCTCCAGTGGAGGAAGGACCAACTACGCCGATAGT SEQ ID GTGAAGGGC NO:287 FR3 AGATTCACCATCTCCAGGGACAACGCCAAGAACACAGTGTAT SEQ ID CTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTTTAT NO:288 TACTGCGCAGCC CDR3 GCCGTGGCTGTGTGCGGAGGATCTTGGTCTTGGTACAACCAG SEQ ID NO:289 FR4 TGGGGGCAGGGCACCCAGGTTACAGTTTCCTCC SEQ ID

[0800] NO:290

[0801]

[0802] CD33-B03 VHH - Amino Acids (SEQ ID NO:291):

[0803] ELQLVESGGGLVQPGGSLRLSCAASGNICGFYAMGWYRQAPGKQRELVAAVAGGGITNYADSVKGRFTVS RDNATNTVYLQMNSLKPEDTAVYYCYADCPNPLLMTMSYFWGQGTQVTVSS (121 aa)

[0804] CD33-B03 VHH - Nucleotides (SEQ ID NO:292):

[0805] GAACTGCAGCTGGTGGAATCCGGCGGAGGATTGGTGCAGCCTGGAGGATCTTTGAGGCTGAGCTGCGCCG CTTCCGGAAACATCTGCGGATTTTATGCCATGGGATGGTACAGGCAGGCCCCTGGAAAACAGAGGGAGCT GGTGGCTGCTGTGGCCGGAGGAGGAATTACCAACTACGCCGACTCCGTGAAGGGAAGGTTCACCGTGAGC C GG GATAAC GCTACTAAC AC C GTGTATCTGC AGATGAAC AG C CTGAAAC CTGAGGATACTG C C GTGTACT ACTGCTACGCCGACTGTCCCAACCCTCTGCTGATGACCATGTCTTACTTCTGGGGCCAGGGCACCCAGGT GACAGTTTCTTCC (363 bp)

[0806] CD33-B03 VHH

[0807] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO:291

[0808] FR1 ELQLVESGGGLVQPGGSLRLSCAASGNICG 1 - 30 30 SEQ ID NO:293 CDR1 FYAMG 31 - 35 5 SEQ ID NO:294 FR2 WYRQAPGKQRELVA 36 - 49 14 SEQ ID NO:295 CDR2 AVAGGGITNYADSVKG 50 - 65 16 SEQ ID

[0809] NO:296

[0810]

[0811] FR3 RFTVSRDNATNTVYLQMNSLKPEDTAVYYCYA 66 - 97 32 SEQ ID NO:297 CDR3 DCPNPLLMTMSYF 98 - 110 13 SEQ ID NO:298 FR4 WGQGTQVTVSS 111 - 11 SEQ ID 121 NO:299

[0812]

[0813] 121

[0814] CD33-B03 VHH

[0815] Region DNA sequence SEQ ID NO FR1 GAACTGCAGCTGGTGGAATCCGGCGGAGGATTGGTGCAGC SEQ ID CTGGAGGATCTTTGAGGCTGAGCTGCGCCGCTTCCGGAAAC NO:300 ATCTGCGGA CDR1 TTTTATGCCATGGGA SEQ ID NO:301 FR2 TGGTACAGGCAGGCCCCTGGAAAACAGAGGGAGCTGGTGG SEQ ID CT NO:302 CDR2 GCTGTGGCCGGAGGAGGAATTACCAACTACGCCGACTCCGT SEQ ID GAAGGGA NO:303 FR3 AGGTTCACCGTGAGCCGGGATAACGCTACTAACACCGTGTA SEQ ID TCTGCAGATGAACAGCCTGAAACCTGAGGATACTGCCGTGT NO:304 ACTACTGCTACGCC CDR3 GACTGTCCCAACCCTCTGCTGATGACCATGTCTTACTTC SEQ ID NO:305 FR4 TGGGGCCAGGGCACCCAGGTGACAGTTTCTTCC SEQ ID

[0816] NO:306

[0817]

[0818] CD33-B04 VHH - Amino Acids (SEQ ID NO:307):

[0819] QVQLVESGGGLVQPGGSLRLSCAASGSISSIYAMGWYRGEQRELVAAILSGGSTKYADSAKGRFTISRDN ATNTVYLQMNSLKPEDTAVYYCNADTVAQDNMVYYWGQGTQVTVSS (116 aa)

[0820] CD33-B04 VHH - Nucleotides (SEQ ID NO:308):

[0821] CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGCCAGGAGGATCACTCAGGCTGTCATGCGCCG CTTCCGGAAGTATCTCCTCCATCTACGCCATGGGCTGGTACAGGGGCGAGCAGAGAGAGTTGGTGGCTGCTATCTTGTCCGGCGGCAGTACCAAGTACGCCGACTCTGCTAAGGGAAGGTTTACAATCAGCAGGGACAAC GCCACCAACACAGTGTACCTGCAGATGAACAGCCTGAAGCCTGAAGACACCGCCGTCTACTATTGCAACG CCGACACCGTGGCCCAGGACAATATGGTGTACTACTGGGGCCAGGGCACCCAGGTTACCGTTTCTTCC (348 bp)

[0822] CD33-B04 VHH

[0823] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO:307

[0824] FR1 QVQLVESGGGLVQPGGSLRLSCAASGSISS 1 - 30 30 SEQ ID NO:309 CDR1 IYAMG 31 - 35 5 SEQ ID NO:310 FR2 WYRGEQRELVA 36 - 49 14 SEQ ID NO:311 CDR2 AILSGGSTKYADSAKG 50 - 65 16 SEQ ID NO:312 FR3 RFTISRDNATNTVYLQMNSLKPEDTAVYYCNA 66 - 94 29 SEQ ID NO:313 CDR3 DTVAQDNMVYY 95 - 105 11 SEQ ID NO:314 FR4 WGQGTQVTVSS 106 - 11 SEQ ID 116 NO:315

[0825]

[0826] 116

[0827] CD33-B04 VHH

[0828] Region DNA sequence SEQ ID NO FR1 CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGC SEQ ID CAGGAGGATCACTCAGGCTGTCATGCGCCGCTTCCGGAAGT NO:316 ATCTCCTCC CDR1 ATCTACGCCATGGGC SEQ ID

[0829] NO:317

[0830]

[0831] FR2 TGGTACAGGGGCGAGCAGAGAGAGTTGGTGGCTGCTATCTT SEQ ID G NO:318 CDR2 TCCGGCGGCAGTACCAAGTACGCCGACTCTGCTAAGGGAA SEQ ID GGTTTACA NO:319 FR3 ATCAGCAGGGACAACGCCACCAACACAGTGTACCTGCAGA SEQ ID TGAACAGCCTGAAGCCTGAAGACACCGCCGTCTACTATTGC NO:320 AACGCC CDR3 GACACCGTGGCCCAGGACAATATGGTGTACTAC SEQ ID NO:321 FR4 TGGGGCCAGGGCACCCAGGTTACCGTTTCTTCC SEQ ID

[0832] NO:322

[0833]

[0834] CD33-B05 VHH - Amino Acids (SEQ ID NO:323):

[0835] QVQLVESGGGLVQPGGSLRLSCAASGSIFSIYGMGWYRQAPGNQRDMVAHITSSGITNYADSVKGRFTIS RDNASNTVYLQMNSLKPEDTAVYYCNADVVGEVPTMGEVPTMHYYWGQGTQVTVSS (126 aa)

[0836] CD33-B05 VHH - Nucleotides (SEQ ID NO:324):

[0837] CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGCCAGGAGGATCACTCAGGCTGTCATGCGCCG CTTCCGGAAGTATCTTCTCCATCTACGGCATGGGCTGGTACAGGCAGGCCCCTGGAAATCAGAGGGACAT GGTGGCTCATATTACCAGTAGCGGAATCACCAACTACGCCGACTCCGTTAAGGGGAGGTTCACTATCAGC AGGGACAACGCTAGCAACACAGTGTATCTGCAGATGAACAGCCTGAAGCCCGAAGACACCGCCGTGTACT ACTGCAACGCAGACGTGGTGGGCGAGGTGCCTACAATGGGAGAGGTGCCAACAATGCACTACTACTGGGG GCAGGGCACCCAGGTTACCGTTTCTTCC (378 bp)

[0838] CD33-B05 VHH

[0839] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO:323

[0840] FR1 QVQLVESGGGLVQPGGSLRLSCAASGSIFS 1 - 30 30 SEQ ID NO:325 CDR1 IYGMG 31 - 35 5 SEQ ID NO:326 FR2 WYRQAPGNQRDMVA 36 - 49 14 SEQ ID

[0841] NO:327

[0842]

[0843] CDR2 HITSSGITNYADSVKG 50 - 65 16 SEQ ID NO:328 FR3 RFTISRDNASNTVYLQMNSLKPEDTAVYYCNA 66 - 97 32 SEQ ID NO:329 CDR3 DVVGEVPTMGEVPTMHYY 98 - 115 18 SEQ ID NO:330 FR4 WGQGTQVTVSS 116 - 11 SEQ ID 126 NO:331

[0844]

[0845] 126

[0846] CD33-B05 VHH

[0847] Region DNA sequence SEQ ID NO FR1 CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGCCA SEQ ID GGAGGATCACTCAGGCTGTCATGCGCCGCTTCCGGAAGTATCT NO:332 TCTCC CDR1 ATCTACGGCATGGGC SEQ ID NO:333 FR2 TGGTACAGGCAGGCCCCTGGAAATCAGAGGGACATGGTGGCT SEQ ID NO:334 CDR2 CATATTACCAGTAGCGGAATCACCAACTACGCCGACTCCGTTA SEQ ID AGGGG NO:335 FR3 AGGTTCACTATCAGCAGGGACAACGCTAGCAACACAGTGTATC SEQ ID TGCAGATGAACAGCCTGAAGCCCGAAGACACCGCCGTGTACT NO:336 ACTGCAACGCA CDR3 GACGTGGTGGGCGAGGTGCCTACAATGGGAGAGGTGCCAACA SEQ ID ATGCACTACTAC NO:337 FR4 TGGGGGCAGGGCACCCAGGTTACCGTTTCTTCC SEQ ID

[0848] NO:338

[0849]

[0850] CD33-B06 VHH - Amino Acids (SEQ ID NO:339):

[0851] QVHLVESGGGLVEPGGSLRLSCAASGSIFHIYFMGWYRQAPGKQRELVAHIFSGGSTNYADSVKGRFTIS RDNAENTVYLQMNSLKFEDTAVYICNMGGSWGRGTQVTVSS (111 aa)

[0852] CD33-B06 VHH - Nucleotides (SEQ ID NO:340):CAGGTTCACCTCGTGGAGTCCGGAGGAGGACTCGTTGAGCCAGGAGGAAGCCTCAGGCTCTCCTGTGCAG CATCCGGAAGCATCTTCCACATCTACTTCATGGGCTGGTACAGACAGGCTCCCGGCAAACAGAGGGAGCT GGTTGCTCATATTTTCAGTGGGGGATCAACCAACTACGCTGATTCCGTTAAGGGCCGATTCACCATCTCC C GG GAC AAC GCTG AG AACAC C GTCTAC CTC C AG ATG AACTC C CTG AAATTC GAAG AC AC CG C C GTGTAC A TCTGCAACATGGGCGGCTCCTGGGGGAGAGGAACACAGGTTACAGTGAGCTCC (333 bp)

[0853] CD33-B06 VHH

[0854] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO:339

[0855] FR1 QVHLVESGGGLVEPGGSLRLSCAASGSIFH 1 - 30 30 SEQ ID NO:341 CDR1 IYFMG 31 - 35 5 SEQ ID NO:342 FR2 WYRQAPGKQRELVA 36 - 49 14 SEQ ID NO:343 CDR2 HIFSGGSTNYADSVKG 50 - 65 16 SEQ ID NO:344 FR3 RFTISRDNAENTVYLQMNSLKFEDTAVYICNM 66 - 97 32 SEQ ID NO:345 CDR3 GGS 98 - 100 3 SEQ ID NO:346 FR4 WGRGTQVTVSS 101 - 11 SEQ ID 111 NO:347

[0856]

[0857] 111

[0858] CD33-B06 VHH

[0859] Region DNA sequence SEQ ID NO FR1 CAGGTTCACCTCGTGGAGTCCGGAGGAGGACTCGTTGAGC SEQ ID CAGGAGGAAGCCTCAGGCTCTCCTGTGCAGCATCCGGAAG NO:348 CATCTTCCAC CDR1 ATCTACTTCATGGGC SEQ ID

[0860] NO:349

[0861]

[0862] FR2 TGGTACAGACAGGCTCCCGGCAAACAGAGGGAGCTGGTT SEQ ID GCT NO:350 CDR2 CATATTTTCAGTGGGGGATCAACCAACTACGCTGATTCCGT SEQ ID TAAGGGC NO:351 FR3 CGATTCACCATCTCCCGGGACAACGCTGAGAACACCGTCT SEQ ID ACCTCCAGATGAACTCCCTGAAATTCGAAGACACCGCCGT NO:352 GTACATCTGCAACATG CDR3 GGCGGCTCC SEQ ID NO:353 FR4 GGCGGCTCCTGGGGGAGAGGAACACAGGTTACAGTGAGC SEQ ID TCC NO:354

[0863]

[0864] CD33-B07 VHH - Amino Acids (SEQ ID NO:355):

[0865] QLQLVESGGGLAQPGGSLRLSCAASGGIFRLYTMGWYRQAPGKQRELVGSISTRGDTNYANSIAGRFSIS RDGDENTVYLQMNSLKSEDTAVYYCSARARPFSTQEDYWGQGTQVTVSS (119 aa)

[0866] CD33-B07 VHH - Nucleotides (SEQ ID NO:356):

[0867] CAGTTGCAGCTCGTGGAGTCCGGAGGAGGACTCGCTCAGCCTGGAGGAAGTCTCAGGCTCTCCTGCGCAG CATCCGGAGGAATCTTCAGGCTCTACACCATGGGCTGGTACAGGCAGGCCCCTGGAAAACAGAGGGAGTT GGTGGGAAGCATTTCCACTAGGGGAGACACCAACTACGCTAACAGTATTGCCGGGAGGTTCTCCATCTCC AGGGACGGAGATGAGAATACAGTCTACCTGCAGATGAACAGCCTGAAGAGTGAGGACACCGCCGTGTACT ATTGCTCCGCTAGGGCCAGACCCTTCTCCACACAGGAGGACTACTGGGGCCAGGGAACCCAGGTTACCGT GTCTTCC (357 bp)

[0868] CD33-B07 VHH

[0869] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO:355

[0870] FR1 QLQLVESGGGLAQPGGSLRLSCAASGGIFR 1 - 30 30 SEQ ID NO:357 CDR1 LYTMG 31 - 35 5 SEQ ID NO:358 FR2 WYRQAPGKQRELVG 36 - 49 14 SEQ ID

[0871] NO:359

[0872]

[0873] CDR2 SISTRGDTNYANSIAG 50 - 65 16 SEQ ID NO:360 FR3 RFSISRDGDENTVYLQMNSLKSEDTAVYYCSA 66 - 97 32 SEQ ID NO:361 CDR3 RARPFSTQEDY 98 - 108 11 SEQ ID NO:362 FR4 WGQGTQVTVSS 109 - 11 SEQ ID 119 NO:363

[0874]

[0875] 119

[0876] CD33-B07 VHH

[0877] Region DNA sequence SEQ ID NO FR1 CAGTTGCAGCTCGTGGAGTCCGGAGGAGGACTCGCTCAGCC SEQ ID TGGAGGAAGTCTCAGGCTCTCCTGCGCAGCATCCGGAGGAAT NO:364 CTTCAGG CDR1 CTCTACACCATGGGC SEQ ID NO:365 FR2 TGGTACAGGCAGGCCCCTGGAAAACAGAGGGAGTTGGTGGG SEQ ID A NO:366 CDR2 AGCATTTCCACTAGGGGAGACACCAACTACGCTAACAGTATT SEQ ID GCCGGG NO:367 FR3 AGGTTCTCCATCTCCAGGGACGGAGATGAGAATACAGTCTAC SEQ ID CTGCAGATGAACAGCCTGAAGAGTGAGGACACCGCCGTGTA NO:368 CTATTGCTCCGCT CDR3 AGGGCCAGACCCTTCTCCACACAGGAGGACTAC SEQ ID NO:369 FR4 TGGGGCCAGGGAACCCAGGTTACCGTGTCTTCC SEQ ID

[0878] NO:370

[0879]

[0880] CD33-B08 VHH - Amino Acids (SEQ ID NO:371):

[0881] QVQLVESGGGLVQAGGSLRLSCAASGSIFAPDVMAWYRQAPGKQREAVAEITSGGLITYVNNIEGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCRYTTAQRLGVMPYDYWGQGTQVTVSS (121 aa)

[0882] CD33-B08 VHH - Nucleotides (SEQ ID NO:372):CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGGCAGGAGGATCACTCAGGCTCTCCTGCGCAG CATCAGGCAGTATCTTCGCCCCCGACGTCATGGCATGGTACAGGCAGGCACCCGGAAAACAGAGGGAGGC TGTGGCTGAAATTACCAGTGGAGGGCTGATCACCTACGTTAACAACATTGAGGGAAGGTTCACTATCTCC AGGGATAACGCCAAGAACACAGTGTATCTGCAGATGAACAGCCTGAAGCCCGAAGACACCGCAGTGTACT ACTGCAGGTACACCACCGCACAGAGGCTGGGAGTGATGCCATACGACTACTGGGGCCAGGGAACCCAGGT GACAGTGAGTTCC (363 bp)

[0883] CD33-B08 VHH

[0884] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO:371

[0885] FR1 QVQLVESGGGLVQAGGSLRLSCAASGSIFA 1 - 30 30 SEQ ID NO:373 CDR1 PDVMA 31 - 35 5 SEQ ID NO:374 FR2 WYRQAPGKQREAVA 36 - 49 14 SEQ ID NO:375 CDR2 EITSGGLITYVNNIEG 50 - 65 16 SEQ ID NO:376 FR3 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCRY 66 - 97 32 SEQ ID NO:377 CDR3 TTAQRLGVMPYDY 98 - 110 13 SEQ ID NO:378 FR4 WGQGTQVTVSS 111 - 11 SEQ ID 121 NO:379

[0886]

[0887] 121

[0888] CD33-B08 VHH

[0889] Region DNA sequence SEQ ID NO FR1 CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGGCA SEQ ID GGAGGATCACTCAGGCTCTCCTGCGCAGCATCAGGCAGTATCT NO:380

[0890]

[0891] TCGCCCDR1 CCCGACGTCATGGCA SEQ ID NO:381 FR2 TGGTACAGGCAGGCACCCGGAAAACAGAGGGAGGCTGTGGC SEQ ID T NO:382 CDR2 GAAATTACCAGTGGAGGGCTGATCACCTACGTTAACAACATTG SEQ ID AGGGA NO:383 FR3 AGGTTCACTATCTCCAGGGATAACGCCAAGAACACAGTGTATC SEQ ID TGCAGATGAACAGCCTGAAGCCCGAAGACACCGCAGTGTACT NO:384 ACTGCAGGTAC CDR3 ACCACCGCACAGAGGCTGGGAGTGATGCCATACGACTAC SEQ ID NO:385 FR4 TGGGGCCAGGGAACCCAGGTGACAGTGAGTTCC SEQ ID

[0892] NO:386

[0893]

[0894] CD33-B09 VHH - Amino Acids (SEQ ID NO:387):

[0895] QVQLVESGGGLVQPGGSLRLSCAASGTMFSLGMVGWYRQAPGNQRELVASSLNGNSSNYVTSVLGRFTLS RDNAKNTVYLQMNSLKPEDTAVYYCNAWVTQRGSVRNYWGQGTQVTVSS (119 aa)

[0896] CD33-B09 VHH - Nucleotides (SEQ ID NO:388):

[0897] CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGCCAGGAGGATCACTCAGGCTGTCATGCGCCG CTTCCGGAACAATGTTCTCCCTCGGCATGGTCGGATGGTACAGACAGGCACCCGGCAATCAGAGGGAGCT GGTTGCTAGCTCCCTGAACGGAAACTCCTCCAACTACGTGACTAGTGTGCTCGGCAGATTCACCCTGTCT AGGGACAACGCCAAGAACACAGTCTATCTCCAGATGAACAGCCTGAAACCCGAAGACACCGCCGTGTACT ACTGCAACGCCTGGGTGACCCAGAGGGGAAGTGTGAGAAACTACTGGGGACAGGGAACCCAGGTCACAGT GTCCTCC (357 bp)

[0898] CD33-B09 VHH

[0899] Region Amino Acid Sequence Residues Length SEQ ID of SEQ NO ID NO:387

[0900] FR1 QVQLVESGGGLVQPGGSLRLSCAASGTMFS 1 - 30 30 SEQ ID NO:389 CDR1 LGMVG 31 - 35 5 SEQ ID

[0901] NO:390

[0902]

[0903] FR2 WYRQAPGNQRELVA 36 - 49 14 SEQ ID NO:391 CDR2 SSLNGNSSNYVTSVLG 50 - 65 16 SEQ ID NO:392 FR3 RFTLSRDNAKNTVYLQMNSLKPEDTAVYYCNA 66 - 97 32 SEQ ID NO:393 CDR3 WVTQRGSVRNY 98 - 108 11 SEQ ID NO:394 FR4 WGQGTQVTVSS 109 - 11 SEQ ID 119 NO:395

[0904]

[0905] 119

[0906] CD33-B09 VHH

[0907] Region DNA sequence SEQ ID NO FR1 CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGC SEQ ID CAGGAGGATCACTCAGGCTGTCATGCGCCGCTTCCGGAACA NO:396 ATGTTCTCC CDR1 CTCGGCATGGTCGGA SEQ ID NO:397 FR2 TGGTACAGACAGGCACCCGGCAATCAGAGGGAGCTGGTTG SEQ ID CT NO:398 CDR2 AGCTCCCTGAACGGAAACTCCTCCAACTACGTGACTAGTGT SEQ ID GCTCGGC NO:399 FR3 AGATTCACCCTGTCTAGGGACAACGCCAAGAACACAGTCTA SEQ ID TCTCCAGATGAACAGCCTGAAACCCGAAGACACCGCCGTG N0:400 TACTACTGCAACGCC CDR3 TGGGTGACCCAGAGGGGAAGTGTGAGAAACTAC SEQ ID NO:401 FR4 TGGGGACAGGGAACCCAGGTCACAGTGTCCTCC SEQ ID

[0908] NO:402

[0909]

[0910] CD33-B10 VHH - Amino Acids (SEQ ID NO:403):

[0911] QVQLVESGGGLVQPGGSLRLSCAASGDIFSIYAMGWYRGKERELVAAITSGGSTNYADSVKGRFTISRDNATNTVYLQMNSLKPEDTDVYYCNADTVAAGNMVYYWGQGTQVTVSS (116 aa)

[0912] CD33-B10 VHH - Nucleotides (SEQ ID NO:404):

[0913] CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGCCAGGAGGATCACTCAGGCTGTCATGCGCCG CTTCCGGAGATATCTTCTCCATCTACGCCATGGGCTGGTACAGAGGCAAAGAGAGGGAACTGGTGGCTGC TATTACCTCCGGCGGCTCAACCAACTACGCCGATTCTGTGAAGGGAAGGTTTACAATTTCCAGGGACAAC GC C AC C AAC ACC GTGTACCTG C AG ATG AATAGTCTGAAAC C C GAAG ATAC C GAG GTCTACTACTG C AAC G CCGACACCGTGGCCGCTGGAAATATGGTGTACTACTGGGGACAGGGAACCCAGGTCACCGTGAGTTCC (348 bp)

[0914] CD33-B10 VHH

[0915] Region Amino Acid Sequence Residues Length SEQ ID of SEQ No: ID NO:403

[0916] FR1 QVQLVESGGGLVQPGGSLRLSCAASGDIFS 1 - 30 30 SEQ ID NO:405 CDR1 IYAMG 31 - 35 5 SEQ ID NO:406 FR2 WYRGKERELVA 36 - 49 14 SEQ ID NO:407 CDR2 AITSGGSTNYADSVKG 50 - 65 16 SEQ ID NO:408 FR3 RFTISRDNATNTVYLQMNSLKPEDTDVYYCNA 66 - 94 29 SEQ ID NO:409 CDR3 DTVAAGNMVYY 95 - 105 11 SEQ ID NO:410 FR4 WGQGTQVTVSS 106 - 11 SEQ ID 116 NO:411

[0917]

[0918] 116

[0919] CD33-B10 VHH

[0920] Region DNA sequence SEQ ID

[0921] NO

[0922]

[0923] FR1 CAGGTTCAGCTCGTCGAGTCAGGCGGAGGACTCGTTCAGC SEQ ID CAGGAGGATCACTCAGGCTGTCATGCGCCGCTTCCGGAGA NO:412 TATCTTCTCC CDR1 ATCTACGCCATGGGC SEQ ID NO:413 FR2 TGGTACAGAGGCAAAGAGAGGGAACTGGTGGCTGCTATTA SEQ ID CC NO:414 CDR2 TCCGGCGGCTCAACCAACTACGCCGATTCTGTGAAGGGAA SEQ ID GGTTTACA NO:415 FR3 ATTTCCAGGGACAACGCCACCAACACCGTGTACCTGCAGA SEQ ID TGAATAGTCTGAAACCCGAAGATACCGACGTCTACTACTGC NO:416 AACGCC CDR3 GACACCGTGGCCGCTGGAAATATGGTGTACTAC SEQ ID NO:417 FR4 TGGGGACAGGGAACCCAGGTCACCGTGAGTTCC SEQ ID

[0924] NO:418

[0925]

[0926] CD33-B11 VHH - Amino Acids (SEQ ID NO:419):

[0927] EVQLVESGGGLVQPGGSLRLSCAASGSIFSIYSMGWYRQAPGNERELAPGNERELVAGITTGGTTIYADS VKGRFTISRDNATNTLYLQMSSLKPEDTAVYYCNADVVGDVATMHYYWGQGTQVTVSS (128 aa)

[0928] CD33-B11 VHH - Nucleotides (SEQ ID NO:420):

[0929] GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTGCAGCCTGGAGGAAGCCTGAGGCTGAGTTGTGCCG CTAGCGGAAGCATCTTTAGTATCTACTCCATGGGATGGTATAGGCAGGCACCTGGCAACGAGAGGGAATT GGCTCCCGGTAATGAGAGAGAGCTGGTGGCTGGAATTACCACTGGAGGAACAACCATCTACGCCGACTCC GTGAAGGGAAGGTTTACCATCAGCAGAGACAACGCCACAAACACCCTGTACCTCCAGATGTCCTCCCTGA AACCTGAAGACACAGCCGTGTACTACTGCAACGCCGACGTGGTGGGCGATGTGGCTACAATGCACTACTA CTGGGGACAGGGCACCCAGGTCACCGTTTCTTCT (384 bp)

[0930] CD33-B11 VHH

[0931] Region Sequence Fragment Residues Length SEQ ID of SEQ NO ID NO:419

[0932] FR1 EVQLVESGGGLVQPGGSLRLSCAASGSIFS 1 - 30 30 SEQ ID

[0933] NO:421

[0934]

[0935] CDR1 IYSMG 31 - 35 5 SEQ ID NO:422 FR2 WYRQAPGNERELAPGNERELVA 36 - 57 22 SEQ ID NO:423 CDR2 GITTGGTTIYADSVKG 58 - 73 16 SEQ ID NO:424 FR3 RFTISRDNATNTLYLQMSSLKPEDTAVYYCNA 74 - 105 32 SEQ ID NO:425 CDR3 DVVGDVATMHYY 106 - 12 SEQ ID 117 NO:426 FR4 WGQGTQVTVSS 118 - 11 SEQ ID 128 NO:427

[0936]

[0937] 128

[0938] CD33-B11 VHH

[0939] Region DNA sequence SEQ ID NO FR1 GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTGCAG SEQ ID CCTGGAGGAAGCCTGAGGCTGAGTTGTGCCGCTAGCGGA NO:428 AGCATCTTTAGT CDR1 ATCTACTCCATGGGA SEQ ID NO:429 FR2 TGGTATAGGCAGGCACCTGGCAACGAGAGGGAATTGGCT SEQ ID CCCGGTAATGAGAGAGAGCTGGTGGCT NO:430 CDR2 GGAATTACCACTGGAGGAACAACCATCTACGCCGACTCCG SEQ ID TGAAGGGA NO:431 FR3 AGGTTTACCATCAGCAGAGACAACGCCACAAACACCCTG SEQ ID TACCTCCAGATGTCCTCCCTGAAACCTGAAGACACAGCCG NO:432 TGTACTACTGCAACGCC CDR3 GACGTGGTGGGCGATGTGGCTACAATGCACTACTAC SEQ ID NO:433 FR4 TGGGGACAGGGCACCCAGGTCACCGTTTCTTCT SEQ ID

[0940] NO:434

[0941]

[0942] (V) Anti-CD33 mono-CARs

[0943] CD33-B01 CAR (without signal peptide) - Amino Acids (SEQ ID NO:435):

[0944] EVQLVESGGGLVQAGGSLRLTCVASGSLLRFGAIGWHRQGPEKQRALVASITDGHTNYADSVKGRFTISR DNAKNTVYLQMNNLKTEDTGIYYCSGATLSLNLRWKYDYWGQGIQVTVSSFVPVFLPAKPTTTPAPRPPT PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (353 aa)

[0945] CD33-B01 CAR (without signal peptide) - Nucleotides (SEQ ID NO:436):

[0946] GAAGTGCAGCTGGTGGAGAGCGGAGGAGGATTGGTGCAGGCAGGAGGAAGCCTGAGGCTGACATGTGTGG CCAGCGGAAGCCTGCTGAGGTTTGGAGCAATCGGATGGCACAGGCAGGGACCTGAGAAGCAGAGGGCACT GGTGGCTAGCATCACCGATGGCCATACCAATTACGCCGACTCCGTGAAAGGAAGGTTCACCATCTCCAGG GACAACGCCAAGAACACCGTGTACTTGCAGATGAACAACCTGAAGACCGAGGACACAGGCATCTACTACT GCTCCGGCGCCACCTTGAGCCTGAACTTGCGATGGAAGTACGACTACTGGGGCCAGGGAATCCAGGTGAC CGTGTCTTCCTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACA CCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGC G CAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGG GGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTC AAAC AAC C ATTTATGAGAC CAGTAC AAACT...

Claims

CLAIMS1. A nucleic acid comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a first polypeptide, wherein the first polypeptide comprises an anti-CLL- 1 antigen-binding region comprising a first VHH, wherein the first VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3. having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO:118, SEQ ID NO: 120, SEQ ID NO: 122, respectively; (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively; (4) SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, respectively; (5) SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, respectively; (6) SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, respectively; (7) SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, respectively; (8) SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, respectively; (9) SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, respectively; (10) SEQ ID NO:

166. SEQ ID NO:168, SEQ ID NO:170, respectively; (11) SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, respectively; or (12) SEQ ID NO:198, SEQ ID N0:200, SEQ ID NO:202, respectively;and wherein the second polynucleotide encodes a second polypeptide, wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:

344. SEQ ID NO:

346. respectively; (3) SEQ ID NO:

422. SEQ ID NO:424, SEQ ID NO:426, respectively; (4) SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, respectively; (5) SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, respectively; (6) SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, respectively; (7) SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, respectively; (8) SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, respectively; (9) SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, respectively; (10) SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, respectively; or (11) SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, respectively.

2. A nucleic acid comprising a first polynucleotide and a second polynucleotide,wherein the first polynucleotide encodes a first polypeptide, wherein the first polypeptide comprises an anti-CLL- 1 antigen-binding region comprising a first VHH, wherein the first VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO:118, SEQ ID NO: 120, SEQ ID NO: 122, respectively; (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively; (4) SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, respectively; (5) SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, respectively; (6) SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, respectively; (7) SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, respectively; (8) SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, respectively; (9) SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, respectively; (10) SEQ ID NO:

166. SEQ ID NO:168, SEQ ID NO:170, respectively; (11) SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, respectively; or (12) SEQ ID NO:198, SEQ ID N0:200, SEQ ID NO:202, respectively;and wherein the second polynucleotide encodes a second polypeptide, wherein the second polypeptide comprises an anti-CD33 antigen-binding region.

3. The nucleic acid of claim 2, wherein the anti-CD33 antigen-binding region comprises a second VHH, wherein the second VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; (3) SEQ ID NO:422, SEQ ID NO:

424. SEQ ID NO:

426. respectively; (4) SEQ ID NO:

262. SEQ ID NO:264, SEQ ID NO:266, respectively; (5) SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, respectively; (6) SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, respectively; (7) SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, respectively; (8) SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, respectively; (9) SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, respectively; (10) SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, respectively; or (11) SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, respectively.

4. A nucleic acid comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a first polypeptide, wherein the first polypeptidecomprises an anti-CLL-1 antigen-binding region;and wherein the second polynucleotide encodes a second polypeptide, wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively; (4) SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, respectively; (5) SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, respectively; (6) SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, respectively; (7) SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, respectively; (8) SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:

362. respectively; (9) SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, respectively; (10) SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, respectively; or (11) SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, respectively.

5. The nucleic acid of claim 4, wherein the anti-CLL-1 antigen-binding region comprises a first VHH, wherein the first VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, respectively; (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively; (4) SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, respectively; (5) SEQ ID NO:

38. SEQ ID NO:40, SEQ ID NO:42, respectively; (6) SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, respectively; (7) SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, respectively; (8) SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, respectively; (9) SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, respectively; (10) SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, respectively; (11) SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, respectively; or (12) SEQ ID NO:198, SEQ ID N0:200, SEQ ID NO:202, respectively.

6. A nucleic acid comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a first polypeptide, wherein the first polypeptidecomprises an anti-CLL- 1 antigen-binding region comprising a first VHH, wherein the first VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:

122. respectively; or (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively;and wherein the second polynucleotide encodes a second polypeptide, wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; or (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively.

7. A nucleic acid comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a first polypeptide, wherein the first polypeptide comprises an anti-CLL- 1 antigen-binding region comprising a first VHH, wherein the first VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, respectively; or (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively;and wherein the second polynucleotide encodes a second polypeptide, wherein the second polypeptide comprises an anti-CD33 antigen-binding region.

8. The nucleic acid of claim 7, wherein the anti-CD33 antigen-binding region comprises a second VHH, wherein the second VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; or (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively.

9. A nucleic acid comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a first polypeptide, wherein the first polypeptide comprises an anti-CLL-1 antigen-binding region;and wherein the second polynucleotide encodes a second polypeptide, wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; or (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively.

10. The nucleic acid of claim 9, wherein the anti-CLL-1 antigen-binding region comprises a first VHH, wherein the first VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO: 118, SEQ ID NO; 120, SEQ ID NO: 122, respectively; or (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively.

11. A nucleic acid comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a first polypeptide, wherein the first polypeptide comprises an anti-CLLl antigen- binding region comprising a first VHH, wherein the first VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, SEQ ID NO: 147, SEQ ID NO: 19, SEQ ID NO:35, SEQ ID NO:

51. SEQ ID NO:83, SEQ ID NO:

99. SEQ ID NO:13L SEQ ID NO:163, SEQ ID NO:179, or SEQ ID NO:195; andwherein the second polynucleotide encodes a second polypeptide, wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:

307. SEQ ID NO:339, SEQ ID NO:419, SEQ ID NO:259, SEQ ID NO:275, SEQ ID NO: 291, SEQ ID NO:323, SEQ ID NO:355, SEQ ID NO:371,SEQ ID NO:387, or SEQ ID NO:403.

12. A nucleic acid comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a first polypeptide, wherein the first polypeptide comprises an anti-CLLl antigen-binding region comprising a first VHH, wherein the first VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, SEQ ID NO: 147, SEQ ID NO: 19, SEQ ID NO:35, SEQ ID NO:51, SEQ ID NO:83, SEQ ID NO:99, SEQ ID NO:

131. SEQ ID NO: 163, SEQ ID NO: 179, or SEQ ID NO: 195; andwherein the second polynucleotide encodes a second polypeptide, wherein the second polypeptide comprises an anti-CD33 antigen-binding region.

13. The nucleic acid of claim 12, wherein the anti-CD33 antigen-binding region comprises a second VHH, wherein the second VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, SEQ ID NO:419, SEQ ID NO:259, SEQ ID NO:275, SEQ ID NO: 291, SEQ ID NO:323, SEQ ID NO:355, SEQ ID NO:371, SEQ ID NO:387, or SEQ ID NO:403.

14. A nucleic acid comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a first polypeptide, wherein the first polypeptide comprises an anti-CLLl antigen-binding region; andwherein the second polynucleotide encodes a second polypeptide, wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:

307. SEQ ID NO:339, SEQ ID NO:419, SEQ ID NO:259, SEQ ID NO:275, SEQ ID NO: 291, SEQ ID NO:323, SEQ ID NO:355, SEQ ID NO:371, SEQ ID NO:387, or SEQ ID NO:403.

15. The nucleic acid of claim 14, wherein the anti-CLLl antigen-binding region comprises a first VHH, wherein the first VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, SEQ ID NO: 147,SEQ ID NO: 19, SEQ ID NO:35, SEQ ID NO:51 , SEQ ID NO:83, SEQ ID NO:99, SEQ ID NO: 131 , SEQ ID NO: 163, SEQ ID NO: 179, or SEQ ID NO: 195.

16. A nucleic acid comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a first polypeptide, wherein the first polypeptide comprises an anti-CLLl antigen- binding region comprising a first VHH, wherein the first VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, or SEQ ID NO: 147; andwherein the second polynucleotide encodes a second polypeptide, wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, or SEQ ID NO:419.

17. A nucleic acid comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a first polypeptide, wherein the first polypeptide comprises an anti-CLLl antigen-binding region comprising a first VHH, wherein the first VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, or SEQ ID NO: 147; andwherein the second polynucleotide encodes a second polypeptide, wherein the second polypeptide comprises an anti-CD33 antigen-binding region.

18. The nucleic acid of claim 17, wherein the anti-CD33 antigen-binding region comprises a second VHH, wherein the second VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, or SEQ ID NO:419.

19. A nucleic acid comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a first polypeptide, wherein the first polypeptide comprises an anti-CLLl antigen-binding region; andwherein the second polynucleotide encodes a second polypeptide, wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, whereinthe second VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, or SEQ ID NO:419.

20. The nucleic acid of claim 19, wherein the anti-CLLl antigen-binding region comprises a first VHH, wherein the first VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, or SEQ ID NO:147.

21. The nucleic acid of any preceding claim, wherein the first polypeptide is a first chimeric antigen receptor (CAR) component, and wherein the second polypeptide is a second CAR component.

22. The nucleic acid of any preceding claim, wherein the first polypeptide further comprises a first hinge region, a first transmembrane domain, or a combination thereof, and wherein the second polypeptide further comprises a second hinge region, a second transmembrane domain, or a combination thereof.

23. The nucleic acid of claim 22, wherein the first hinge region is different from, or the same as, the second hinge region.

24. The nucleic acid of claim 22, wherein the first transmembrane domain is different from, or the same as, the second transmembrane domain.

25. The nucleic acid of any preceding claim, wherein the first polypeptide further comprises a first cytoplasmic signaling domain, and wherein the second polypeptide further comprises a second co-stimulatory region.

26. The nucleic acid of any preceding claim, wherein the first polypeptide further comprises a first co-stimulatory region, and wherein the second polypeptide further comprises a second cytoplasmic signaling domain.

27. The nucleic acid of any preceding claim, wherein the first polynucleotide is located at the 5’ end of the second polynucleotide.

28. The nucleic acid of any of claims 1-26, wherein the second polynucleotide is located at the 5’ end of the first polynucleotide.

29. The nucleic acid of any preceding claim, wherein the first polypeptide comprises, from N-terminus to C-terminus: the first VHH, a first hinge region, a first transmembrane domain, and a first cytoplasmic signaling domain.

30. The nucleic acid of any preceding claim, wherein the second polypeptide comprises, from N-terminus to C-terminus: the second VHH, a second hinge region, a second transmembrane domain, and a second co- stimulatory region.

31. The nucleic acid of any preceding claim, wherein the first polypeptide and the second polynucleotide are linked by a linker.

32. The nucleic acid of claim 31, wherein the linker is a cleavable linker.

33. The nucleic acid of any preceding claim, wherein the first polypeptide further comprises a first signal peptide, and wherein the second polypeptide further comprises a second signal peptide.

34. The nucleic acid of claim 33, wherein the first signal peptide is the same as, or different from, the second signal peptide.

35. The nucleic acid of any preceding claim, wherein the first polynucleotide comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:572, SEQ ID NO:574, SEQ ID NO:532, SEQ ID NO:534, SEQ ID NO: 482, SEQ ID NO:484, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:502, SEQ ID NO:504, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:

522. SEQ ID NO:524, SEQ ID NO:542, SEQ ID NO:544, SEQ ID NO:552, SEQ ID NO:554, SEQ ID NO:562, or SEQ ID NO:564.

36. The nucleic acid of any preceding claim, wherein the second polynucleotide comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:576, SEQ ID NO:578, SEQ ID NO:536, SEQ ID NO:538, SEQ ID NO:486, SEQ ID NO:488, SEQ ID NO:496, SEQ ID NO:498, SEQ ID NO:506, SEQ ID NO:508, SEQ ID NO:516, SEQ ID NO:518, SEQ ID NO:

526. SEQ ID NO:

528. SEQ ID NO:546, SEQ ID NO:548, SEQ ID NO:556, SEQ ID NO:558, SEQ ID NO:566, or SEQ ID NO:568.

37. The nucleic acid of any preceding claim, wherein the first polypeptide comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:571, SEQ ID NO:573, SEQ ID NO:

531. SEQ ID NO:533, SEQ ID NO:481, SEQ ID NO:483, SEQ ID NO:491, SEQ ID NO:493, SEQ ID NO:501, SEQ ID NO:503, SEQ ID NO:511, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:523, SEQ ID NO:541, SEQ ID NO:543, SEQ ID NO:551, SEQ ID NO:553, SEQ ID NO:

561. or SEQ ID NO:563.

38. The nucleic acid of any preceding claim, wherein the second polypeptide comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:575, SEQ ID NO:577, SEQ ID NO:535, SEQ ID NO:537, SEQ ID NO:485, SEQ ID NO:487, SEQ ID NO:495, SEQ ID NO:497, SEQ ID NO:505, SEQ ID NO:507, SEQ ID NO:515, SEQ ID NO:517, SEQ ID NO:525, SEQ ID NO:527, SEQ ID NO:545, SEQ ID NO:547, SEQ ID NO:555, SEQ ID NO:557, SEQ ID NO:565, or SEQ ID NO:567.

39. The nucleic acid of any preceding claim, wherein the nucleic acid comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:570, SEQ ID NO:530, SEQ ID NO:

480. SEQ ID NO:490, SEQ ID NO:500, SEQ ID NO:510, SEQ ID NO:520, SEQ ID NO:540, SEQ ID NO:550, SEQ ID NO:560.

40. The nucleic acid of any preceding claim, encoding a polypeptide comprising an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:569, SEQ ID NO:529, SEQ ID NO:

479. SEQ ID NO:489, SEQ ID NO:499, SEQ ID NO:509, SEQ ID NO:519, SEQ ID NO:539, SEQ ID NO:549, or SEQ ID NO:559.

41. The nucleic acid of any of claims 1-20, 27, 28, 31 and 32, encoding a bispecific antibody, wherein the first polypeptide is a first antibody fragment, and wherein the second polypeptide is a second antibody fragment.

42. A recombinant polypeptide, comprising an anti-CLL-1 VHH, wherein the anti-CLL-1 VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 comprises amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, respectively; (3) SEQ ID NO: 150, SEQ ID NO:

152. SEQ ID NO: 154, respectively; (4) SEQ ID NO;22, SEQ ID NO:24, SEQ ID NO:26, respectively; (5) SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, respectively; (6) SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, respectively; (7) SEQ ID NO:

86. SEQ ID NO:

88. SEQ ID NO;90. respectively; (8) SEQ ID NO: 102, SEQ ID NO:

104. SEQ ID NO: 106, respectively; (9) SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, respectively; (10) SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, respectively; (11) SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, respectively; or (12) SEQ ID NO: 198, SEQ ID N0:200, SEQ ID NO:202, respectively.

43. A recombinant polypeptide, comprising an anti-CLL-1 VHH, wherein the anti-CLL-1 VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 comprises amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively: (2) SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, respectively; or (3) SEQ ID NO: 150, SEQ ID NO:

152. SEQ ID NO:

154. respectively.

44. A recombinant polypeptide, comprising an anti-CLL- 1 VHH, wherein the anti-CLL- 1 VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, SEQ ID NO: 147, SEQ ID NO: 19, SEQ ID NO:35, SEQ ID NO:51, SEQ ID NO:83, SEQ ID NO:

99. SEQ ID NO:131, SEQ ID NO:163, SEQ ID NO: 179, or SEQ ID NO: 195.

45. A recombinant polypeptide, comprising an anti-CLL-1 VHH, wherein the anti-CLL-1 VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, or SEQ ID NO: 147.

46. The recombinant polypeptide of any of claims 42-45, comprising an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO: 225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO: 235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO: 245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, or SEQ ID NO:257.

47. A recombinant polypeptide, comprising an anti-CD33 VHH, wherein the anti-CD33 VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 comprises amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; (3) SEQ ID NO:422, SEQ ID NO:

424. SEQ ID NO:426, respectively; (4) SEQ ID NO:

262. SEQ ID NO:264, SEQ ID NO:266, respectively; (5) SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, respectively; (6) SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, respectively; (7) SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, respectively; (8) SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, respectively; (9) SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, respectively; (10) SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, respectively; or (11) SEQ ID NO:406, SEQ ID NO:

408. SEQ ID NO:

410. respectively.

48. A recombinant polypeptide, comprising an anti-CD33 VHH, wherein the anti-CD33 VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 comprises amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ IDNO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; or (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively.

49. A recombinant polypeptide, comprising an anti-CD33 VHH, wherein the anti-CD33 VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:

307. SEQ ID NO;339, SEQ ID NO:419, SEQ ID NO:259, SEQ ID NO:275, SEQ ID NO: 291, SEQ ID NO:323, SEQ ID NO:355, SEQ ID NO:371, SEQ ID NO:387, or SEQ ID NO:403.

50. A recombinant polypeptide, comprising an anti-CD33 VHH, wherein the anti-CD33 VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, or SEQ ID NO:419.

51. The recombinant polypeptide of any of claims 47-50, comprising an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:435, SEQ ID NO:437, SEQ ID NO:439, SEQ ID NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ ID NO:449, SEQ ID NO:451, SEQ ID NO;453, SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459, SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465, SEQ ID NO:467, SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID NO:

475. or SEQ ID NO:477.

52. The recombinant polypeptide of any of claims 42-51 , wherein the recombinant polypeptide is a chimeric antigen receptor (CAR) or an antibody.

53. The nucleic acid of any of claims 1-21, or the recombinant polypeptide of any of claims 42-52, wherein the first polypeptide, the second polypeptide, or the recombinant polypeptide is a chimeric antigen receptor (CAR), and wherein the CAR further comprises one or more of the following:(a) a signal peptide,(b) a hinge region,(c) a transmembrane domain,(d) a co- stimulatory region, and(e) a cytoplasmic signaling domain.

54. The nucleic acid or the recombinant polypeptide of claim 53. wherein the co-stimulatory region comprises a co-stimulatory region of 4- IBB (CD 137), CD28, 0X40, CD2, CD7, CD27, CD30, CD40, CD70, CD 134, PD1, DaplO, CDS, ICAM-1, LFA-1 (CDlla / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

55. The nucleic acid or the recombinant polypeptide of claim 53, wherein the cytoplasmic signaling domain comprises a cytoplasmic signaling domain of CD3i^.

56. The nucleic acid or the recombinant polypeptide of claim 53, wherein the hinge region comprises a hinge region of IgG4, CD8, CD28, CD137, or combinations thereof.

57. The nucleic acid or the recombinant polypeptide of claim 53, wherein the transmembrane domain comprises a transmembrane domain of CD8, CD28, CD3s, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof.

58. The recombinant polypeptide of any of claims 42-52, further comprising a human Fc region.

59. An immune cell comprising the nucleic acid of any of claims 1-41.

60. An immune cell expressing a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an anti-CLL-1 antigen-binding region comprising a first VHH, wherein the first VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively: (2) SEQ ID NO:

118. SEQ ID NO: 120, SEQ ID NO: 122, respectively: (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively; (4) SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, respectively; (5) SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, respectively; (6) SEQ ID NO:

54. SEQ ID NO:

56. SEQ ID NO:58, respectively; (7) SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, respectively; (8) SEQ ID NO: 102, SEQ ID NO: 104,SEQ ID NO: 106, respectively; (9) SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, respectively; (10) SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, respectively; (11) SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:

186. respectively; or (12) SEQ ID NO: 198, SEQ ID N0:200, SEQ ID NO:202, respectively;and wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively; (4) SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, respectively; (5) SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, respectively; (6) SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, respectively; (7) SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, respectively; (8) SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, respectively; (9) SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, respectively; (10) SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, respectively; or (11) SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, respectively.

61. An immune cell expressing a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an anti-CLL-1 antigen-binding region comprising a first VHH, wherein the first VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, respectively; (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively; (4) SEQ ID NO:22, SEQ ID NO:

24. SEQ ID NO:

26. respectively; (5) SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, respectively; (6) SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, respectively; (7) SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:

90. respectively; (8) SEQ ID NO: 102, SEQ ID NO:

104. SEQ ID NO: 106, respectively; (9) SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, respectively; (10) SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, respectively; (11) SEQ ID NO: 182, SEQ ID NO:

184. SEQ ID NO: 186, respectively; or (12) SEQ ID NO: 198, SEQ ID N0:200, SEQ ID NO:202, respectively;and wherein the second polypeptide comprises an anti-CD33 antigen-binding region.

62. The immune cell of claim 61, wherein the anti-CD33 antigen-binding region comprises a second VHH, wherein the second VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; (3) SEQ ID NO:422, SEQ ID NO:

424. SEQ ID NO:426, respectively; (4) SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, respectively; (5) SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, respectively; (6) SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, respectively; (7) SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, respectively; (8) SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, respectively; (9) SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, respectively; (10) SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, respectively; or (11) SEQ ID NO:406, SEQ ID NO:

408. SEQ ID NO;410. respectively.

63. An immune cell expressing a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an anti-CLL-1 antigen-binding region;and wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively; (4) SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, respectively; (5) SEQ ID NO:278, SEQ ID NO:

280. SEQ ID NO:282, respectively; (6) SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:

298. respectively; (7) SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, respectively; (8) SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, respectively; (9) SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, respectively; (10) SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, respectively; or (11) SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, respectively.

64. The immune cell of claim 63, wherein the anti-CLL-1 antigen-binding region comprises afirst VHH, wherein the first VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, respectively; (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively; (4) SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, respectively; (5) SEQ ID NO:

38. SEQ ID NO:40, SEQ ID NO:42, respectively; (6) SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, respectively; (7) SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, respectively; (8) SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, respectively; (9) SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, respectively; (10) SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, respectively; (11) SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, respectively; or (12) SEQ ID NO:198, SEQ ID N0:200, SEQ ID NO:202, respectively.

65. An immune cell expressing a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an anti-CLL-1 antigen-binding region comprising a first VHH, wherein the first VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively: (2) SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, respectively; or (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, respectively;and wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; or (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively.

66. An immune cell expressing a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an anti-CLL-1 antigen-binding region comprising a first VHH, wherein the first VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, respectively; or (3) SEQ ID NO: 150, SEQ ID NO:

152. SEQ ID NO: 154, respectively;and wherein the second polypeptide comprises an anti-CD33 antigen-binding region.

67. The immune cell of claim 66, wherein the anti-CD33 antigen-binding region comprises a second VHH, wherein the second VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, respectively; or (3) SEQ ID NO:422, SEQ ID NO:

424. SEQ ID NO:426, respectively.

68. An immune cell expressing a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an anti-CLL-1 antigen-binding region;and wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, respectively; (2) SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:

346. respectively; or (3) SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, respectively.

69. The immune cell of claim 68. wherein the anti-CLL-1 antigen-binding region comprises a first VHH, wherein the first VHH comprises three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100% identical to the amino acid sequences set forth in (1) SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, respectively; (2) SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, respectively; or (3) SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:

154. respectively.

70. An immune cell expressing a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an anti-CLLl antigen-binding region comprising a first VHH, wherein the first VHH comprises an amino acid sequence about 80% to about 100%identical to the amino acid sequence set forth in SEQ TD NO: 67, SEQ ID NO: 115, SEQ ID NO: 147, SEQ ID NO: 19, SEQ ID NO:35, SEQ ID NO:51, SEQ ID NO:83, SEQ ID NO:99, SEQ ID NO: 131, SEQ ID NO: 163, SEQ ID NO:

179. or SEQ ID NO: 195; andwherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, SEQ ID NO:419, SEQ ID NO:259, SEQ ID NO:275, SEQ ID NO: 291, SEQ ID NO:323, SEQ ID NO:355, SEQ ID NO:371, SEQ ID NO:387, or SEQ ID NO:403.

71. An immune cell expressing a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an anti-CLLl antigen-binding region comprising a first VHH, wherein the first VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, SEQ ID NO: 147, SEQ ID NO: 19, SEQ ID NO:35, SEQ ID NO:51, SEQ ID NO:83, SEQ ID NO:

99. SEQ ID NO: 131, SEQ ID NO: 163, SEQ ID NO: 179, or SEQ ID NO: 195; andwherein the second polypeptide comprises an anti-CD33 antigen-binding region.

72. The immune cell of claim 71, wherein the anti-CD33 antigen-binding region comprises a second VHH, wherein the second VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, SEQ ID NO:419, SEQ ID NO:259, SEQ ID NO:275, SEQ ID NO: 291, SEQ ID NO:323, SEQ ID NO:355, SEQ ID NO:371, SEQ ID NO:387, or SEQ ID NO:403.

73. An immune cell expressing a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an anti-CLLl antigen- binding region; and wherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, SEQ ID NO:419, SEQ ID NO:259, SEQ ID NO:275, SEQ ID NO: 291, SEQ ID NO:323, SEQ ID NO:355, SEQ ID NO:371, SEQ ID NO:387, or SEQ ID NO:403.

74. The immune cell of claim 73, wherein the anti-CLLl antigen-binding region comprises a first VHH, wherein the first VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO:

115. SEQ ID NO: 147, SEQ ID NO: 19, SEQ ID NO:35, SEQ ID NO:51, SEQ ID NO:83, SEQ ID NO:99, SEQ ID NO: 131, SEQ ID NO: 163, SEQ ID NO: 179, or SEQ ID NO: 195.

75. An immune cell expressing a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an anti-CLLl antigen-binding region comprising a first VHH, wherein the first VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, or SEQ ID NO: 147; andwherein the second polypeptide comprises an anti-CD33 antigen-binding region comprising a second VHH, wherein the second VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NO:307, SEQ ID NO:339, or SEQ ID NO:419.

76. An immune cell expressing a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an anti-CLLl antigen-binding region comprising a first VHH, wherein the first VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, or SEQ ID NO: 147; andwherein the second polypeptide comprises an anti-CD33 antigen-binding region.

77. The immune cell of claim 76, wherein the anti-CD33 antigen-binding region comprises a second VHH, wherein the second VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, or SEQ ID NO:419.

78. An immune cell expressing a first polypeptide and a second polypeptide,wherein the first polypeptide comprises an anti-CLLl antigen- binding region; and wherein the second polypeptide comprises an anti-CD33 antigen-binding regioncomprising a second VHH, wherein the second VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO:307, SEQ ID NO:339, or SEQ ID NO:419.

79. The immune cell of claim 78, wherein the anti-CLLl antigen-binding region comprises a first VHH, wherein the first VHH comprises an amino acid sequence about 80% to about 100% identical to the amino acid sequence set forth in SEQ ID NO: 67, SEQ ID NO: 115, or SEQ ID NO:147.

80. An immune cell expressing the recombinant polypeptide of any of claims 42-58.

81. A nucleic acid encoding the recombinant polypeptide of any of claims 42-58.

82. The nucleic acid of claim 81, wherein the nucleic acid comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO: 226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO: 236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO: 246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, or SEQ ID NO:258.

83. The nucleic acid of claim 81, wherein the nucleic acid comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:442, SEQ ID NO:444, SEQ ID NO:

446. SEQ ID NO:448, SEQ ID NO:450, SEQ ID NO:

452. SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458, SEQ ID NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ ID NO:476, or SEQ ID NO:478.

84. A vector comprising the nucleic acid of any of claims 1-41, 53-57 and 81-83.

85. An immune cell comprising the nucleic acid of any of claims 81-83.

86. The immune cell of any of claims 59-80 and 85, wherein the immune cell is a T cell or a natural killer (NK) cell.

87. A pharmaceutical composition, comprising the immune cell of any of claims 59-80, 85 and 86, the nucleic acid of any of claims 1-41, 53-57 and 81-83, the recombinant polypeptide of any of claims 42-58, or the vector of claim 84.

88. A method of treating cancer in a subject, the method comprising administering the immune cell of any of claims 59-80, 85 and 86, or the pharmaceutical composition of claim 87, to the subject.

89. The method of claim 88, wherein the cancer is a hematologic cancer.

90. The method of claim 88, wherein the cancer is Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, leukemia, and / or multiple myeloma.

91. The method of claim 88, wherein the cancer is acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), or combinations thereof.

92. A method of treating an autoimmune disorder in a subject, the method comprising administering the immune cell of any of claims 59-80, 85 and 86, or the pharmaceutical composition of claim 87, to the subject.

93. The method of claim 92, wherein the autoimmune disorder is systemic lupus erythematosus (SLE), systemic sclerosis, inflammatory myopathy, systemic scleroderma, multiple sclerosis, myasthenia gravis, a myositis autoantibody-driven disease, or neuromyelitis optica.

94. The method of claim 93, wherein the inflammatory myopathy is polymyositis, dermatomyositis, or inclusion-body myositis.

95. The method of claim 93, wherein the subject with SLE has lupus nephritis.

96. The method of any of claims 88-95, wherein the immune cell is allogeneic or autologous.

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