FIELD

The present invention relates to CD123-binding polypeptides, and methods of using CD123-binding polypeptides to modulate the biological activity of CD123. Such methods include, but are not limited to, methods of treating cancer.

BACKGROUND

CD123, or interleukin-3 receptor alpha chain (IL-3Rα), has been identified as a leukemia stem cell marker and is often upregulated on AML blasts, while its expression is low or absent on normal hematopoietic stem cells. CD123 may play a role in proliferation, differentiation, and survival of hematopoietic cells. Poor prognosis, high blast counts, and resistance to apoptotic cell death have been associated with high expression CD123 in hematologic cancers, such as leukemia. Therefore, there exists a therapeutic need for more potent treatments for CD123-expressing cancers.

SUMMARY

Provided herein are CD123-binding polypeptides and methods of using CD123-binding polypeptides to treat, for example, leukemia. In some embodiments, a CD123-binding polypeptide comprises at least one VHH domain. Some embodiments are provided below.

Embodiment 1

A polypeptide comprising at least one VHH domain that binds CD123 and that comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 33, 42, 3, 7, 11, 15, 19, 23, 36, 39, 45, 48, 51, or 93; a CDR2 comprising the amino acid sequence of SEQ ID NO: 34, 43, 4, 8, 12, 16, 20, 24, 37, 40, 46, 49, 52, or 94; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 35, 44, 5, 9, 13, 17, 21, 25, 38, 41, 47, 50, 53, or 95.

Embodiment 2

The polypeptide of embodiment 1, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 33 or 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 34 or 4; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 35 or 5.

Embodiment 3

The polypeptide of embodiment 1 or embodiment 2, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 42, 19, or 45; a CDR2 comprising the amino acid sequence of SEQ ID NO: 43, 20, or 46; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 44, 21, or 47.

Embodiment 4

The polypeptide of any one of embodiments 1-3, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7 or 36; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or 37; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9 or 38.

Embodiment 5

The polypeptide of any one of embodiments 1-4, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 15 or 39; a CDR2 comprising the amino acid sequence of SEQ ID NO: 16 or 40; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17 or 41.

Embodiment 6

The polypeptide of any one of embodiments 1-5, wherein at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, 48, 51, or 93; a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, 49, 52, or 94; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25, 50, 53, or 95.

Embodiment 7

The polypeptide of any one of embodiments 1-6, wherein at least one VHH domain comprises a CDR1, a CDR2, and a CDR3, respectively comprising the amino acid sequences of SEQ ID NOs: 33, 34, and 35; 42, 43, and 44; 3, 4, and 5; 7, 8, and 9; 11, 12, and 13; 15, 16, and 17; 19, 20, and 21; 23, 24, and 25; 36, 37, and 38; 39, 40, and 41; 45, 46, and 47; 48, 49, and 50; 51, 52, and 53; or 93, 94, and 95.

Embodiment 8

The polypeptide of any one of embodiments 1-7, wherein at least one VHH domain is humanized.

Embodiment 9

The polypeptide of any one of embodiments 1-8, wherein at least one VHH domain comprises an amino acid sequence at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 32, 26, 27, 28, 29, 30, 31, or 92.

Embodiment 10

The polypeptide of any one of embodiments 1-8, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 32, 26, 27, 28, 29, 30, 31, or 92.

Embodiment 11

The polypeptide of any one of embodiment 1-7, wherein at least one VHH domain comprises an amino acid sequence at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2, 6, 10, 14, 18, or 22.

Embodiment 12

The polypeptide of any one of embodiments 1-7, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 2, 6, 10, 14, 18, or 22.

Embodiment 13

The polypeptide of any one of embodiments 1-12, comprising two VHH domains.

Embodiment 14

The polypeptide of any one of embodiments 1-12, comprising three VHH domains.

Embodiment 15

The polypeptide of any one of embodiments 1-14, wherein the polypeptide comprises at least one binding domain that binds an antigen other than CD123.

Embodiment 16

The polypeptide of embodiment 15, wherein the polypeptide comprises at least one binding domain that binds CD3, T-cell receptor (TCR) α, TCRβ, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D.

Embodiment 17

The polypeptide of embodiment 13 or 14, wherein each VHH domain binds CD123.

Embodiment 18

The polypeptide of embodiment 17, wherein each VHH domain comprises the same CDR1, CDR2, and CDR3 amino acid sequences.

Embodiment 19

The polypeptide of embodiment 17, wherein each VHH domain comprises the same VHH sequence.

Embodiment 20

The polypeptide of any one of embodiments 1-12, comprising one VHH domain.

Embodiment 21

The polypeptide of any one of embodiments 1-20, wherein the polypeptide comprises an Fc region.

Embodiment 22

The polypeptide of embodiment 21, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 54-89.

Embodiment 23

The polypeptide of embodiment 21 or embodiment 22, which forms a dimer under physiological conditions.

Embodiment 24

The polypeptide of any one of embodiments 1-23, wherein the CD123 is human CD123.

Embodiment 25

The polypeptide of embodiment 24, wherein the human CD123 comprises the sequence of SEQ ID NO: 1.

Embodiment 26

The polypeptide of any one of the preceding embodiments, wherein the polypeptide blocks binding of CD123 to IL-3.

Embodiment 27

An immunoconjugate comprising the polypeptide of any one of embodiments 1-26 and a cytotoxic agent.

Embodiment 28

The immunoconjugate of embodiment 31, wherein the cytotoxic agent is selected from a calicheamicin, an auristatin, a dolastatin, a tubulicin, a maytansinoid, a cryptophycin, a duocarmycin, an esperamicin, a pyrrolobenzodiazepine, and an enediyne antibiotic.

Embodiment 29

A pharmaceutical composition comprising the polypeptide of any one of embodiments 1-26 or the immunoconjugate of embodiment 27 or embodiment 28, and a pharmaceutically acceptable carrier.

Embodiment 30

An isolated nucleic acid that encodes the polypeptide of any one of embodiments 1-26.

Embodiment 31

A vector comprising the nucleic acid of embodiment 30.

Embodiment 32

A host cell comprising the nucleic acid of embodiment 30 or the vector of embodiment 31.

Embodiment 33

A host cell that expresses the polypeptide of any one of embodiments 1-26.

Embodiment 34

A method of producing the polypeptide of any one of embodiments 1-26, comprising incubating the host cell of embodiment 32 or embodiment 33 under conditions suitable for expression of the polypeptide.

Embodiment 35

The method of embodiment 34, further comprising isolating the polypeptide.

Embodiment 36

A method of treating cancer comprising administering to a subject with cancer a pharmaceutically effective amount of the polypeptide of any one of embodiments 1-26, the immunoconjugate of embodiment 27 or embodiment 28, or the pharmaceutical composition of embodiment 30.

Embodiment 37

The method of embodiment 36, wherein the cancer is selected from lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); Hairy cell leukemia; and chronic myeloblastic leukemia.

Embodiment 38

The method of embodiment 36 or 37, wherein the cancer is acute myeloid leukemia (AML).

Embodiment 39

The method of any one of embodiments 36-38, further comprising administering an additional therapeutic agent.

Embodiment 40

The method of embodiment 39, wherein the additional therapeutic agent is an anti-cancer agent.

Embodiment 41

The method of embodiment 40, wherein the anti-cancer agent is selected from a chemotherapeutic agent, an anti-cancer biologic, radiation therapy, CAR-T therapy, and an oncolytic virus.

Embodiment 42

The method of any one of embodiments 36-41, wherein the cancer is a CD123-expressing cancer.

