Anti-CD180 binding molecules and uses thereof

Abstract

The present disclosure provides anti-CD180 binding molecules and uses thereof. In one embodiment, the anti-CD180 binding molecule is an anti-CD180 antibody. Also provided is an anti-CD180 antibody-drug conjugate (ADC) comprising a monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors to the cytotoxic drug payload. The anti-CD180 antibody or antigen-binding fragment thereof, and the ADC comprising the anti-CD180 antibody or antigen-binding fragment thereof, are useful in treating diseases such as acute myeloid leukemia, mantle cell lymphoma, multiple myeloma, follicular lymphoma, acute B-lymphoblastic leukemia, or diffuse large B-cell lymphoma.

Description

[Technical Field]

[0001] Incorporation of sequence listings The sequence listing, which follows the rules of WIPO standard ST.26 formatting, is incorporated herein by reference. The sequence listing has been encoded as XML in UTF-8 text and submitted as an electronic document via the Patent Center. The filename of this electronic document, created on January 30, 2024, is "P-620104-USP_ST26.xml" and is 236,802 bytes in size.

[0002] The present disclosure generally relates to the field of antibodies and antibody drug conjugates (ADCs). In one embodiment, the present disclosure provides anti-CD180 binding molecules and uses thereof. In particular, the present disclosure relates to an anti-CD180 antibody drug conjugate (ADC) comprising a monoclonal antibody or antigen-binding fragment thereof that targets a CD180-high-expressing tumor, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets a CD180-high-expressing tumor to the cytotoxic drug payload. [Background technology]

[0003] Toll-like receptors (TLRs) are pattern recognition receptors that bind diverse microbial products, such as microbial membrane lipids or nucleic acids. Antigen-presenting cells (APCs), including B cells, DCs, and macrophages, express multiple TLRs that are bound by pathogens and activate the NFκB and MAP kinase pathways, leading to the expression of costimulatory molecules and cytokine secretion. While TLRs are important for defense against infectious diseases, growing evidence indicates that TLRs also function as regulators of immune responses in cancer, autoimmune diseases, and transplantation.

[0004] CD180 is an orphan member of the TLR family. It is the human homolog of the murine surface receptor RP105, with which it shares 74% sequence identity. The CD180 receptor was first discovered on mouse B cells and identified on human B cells by the Bgp95 mAb. It is a membrane-bound receptor with a molecular weight of 105 kD. The extracellular portion of CD180 consists of tandem repeats of a leucine-rich motif separated from a single transmembrane domain by a carboxy-proximal flanking region that is thought to be involved in processes such as cell adhesion or receptor / ligand interaction. The presence of a conserved cysteine ​​residue in the carboxy-proximal region of CD180 is a feature shared with other TLRs. CD180 is expressed on antigen-presenting cells such as human B lymphocytes, monocytes, and dendritic cells. Histological studies have shown that CD180 is primarily expressed on mature B cells in the mantle zone, whereas its expression in germinal center cells is either very low or negative. CD180 forms a complex with MD-1, which is structurally related to the TLR4 / MD-2 complex, and initiates responses to bacterial LPS.

[0005] CD180 is homologous to TLR4 but lacks its intracellular TLR-like domain and shares 61% sequence similarity in the extracellular domain. These two receptors utilize two distinct signaling pathways: LPS binding to CD180 / MD1 induces Lyn activation and CD19 phosphorylation, whereas LPS binding to TLR4 / MD-2 activates the MyD88 / IRAK and MyD88-independent Toll-interleukin-1 receptor (TIR) ​​domain-containing adaptor protein (TIRAP) pathways, leading to activation of JNK and NFκB.

[0006] Several studies support the importance of CD180 in B cell survival, activation, proliferation, and / or differentiation. Although TLRs possess intracellular TIR domains and the adaptor molecule Myd88, which activates various downstream protein kinases, CD180 lacks a functional cytoplasmic signaling domain and cannot independently transmit intracellular signals. Therefore, it must recruit or cooperate with other receptor pathways. The CD180-mediated signaling pathway is independent of Myd88 expression, and its function is regulated by CD19. In mice, CD180 ligation recruits CD19 to lipid rafts and induces its phosphorylation, which in turn amplifies the activity of the Src kinase Lyn. Phosphorylated CD19 recruits Vav, which is important for upstream activity in the JNK pathway. Thus, CD19 may mediate the interaction between Lyn and Vav in CD180-mediated signaling. On the other hand, activation of PI3K and NFκB by CD180 ligation has been shown to be CD19-independent.

[0007] The contribution of the microenvironment to the pathogenesis of lymphoproliferative disorders (LPDs) is clearly recognized. Next-generation sequencing studies of LPDs have reported impairment of multiple molecules in signaling pathways activated by downstream microenvironmental stimuli, including Toll-like receptor (TLR) signaling. CD180 can modulate the ligand-induced activity of TLR2 and TLR4 and positively regulate CD19 signaling in mouse models. Like many TLRs, CD180 appears to be more highly expressed by memory B cells than naive B cells. Flow cytometry (FCM) analysis of peripheral blood (PB) has reported high CD180 expression by B cells in marginal zone lymphoma (MZL), whereas CD180 expression levels are significantly lower in other LPDs, such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). These results suggest that CD180 may be considered a single positive, reliable marker for MZL and should be included in flow cytometry panels for the diagnosis of mature B-cell neoplasms.

[0008] Accumulating evidence also indicates that CD180 is closely related to the pathogenesis of autoimmune diseases. CD180 can regulate the function of antigen-presenting cells and control the development of collagen-induced arthritis. The CD180-negative B cell population is significantly increased in patients with systemic lupus erythematosus (SLE), which changes depending on the disease activity. Abnormal activation of macrophages and DCs has been shown to contribute to the pathogenesis of SLE. It has been reported that CD180 ligation significantly inhibits TLR7- and TLR9-mediated activation of macrophages and DCs via the Lyn-SHP-1 / 2 pathway, and treatment with anti-CD180 antibodies effectively ameliorated lupus symptoms in lupus-susceptible mice. Taken together, these results indicate that CD180 plays an important role in regulating the activation of the TLR7 and TLR9 pathways in macrophages and DCs, suggesting that CD180 could be utilized as a potential therapeutic target for the treatment of SLE.

[0009] As mentioned above, there is a need for further development of anti-CD180 binding molecules that will facilitate the study and control of CD180-mediated immune responses in B cell and lymphoproliferative disorders. Summary of the Invention

[0010] The present disclosure describes the isolation of a panel of anti-CD180 binding molecules and their uses. In one embodiment, the present disclosure provides an isolated anti-CD180 binding molecule comprising a set of three heavy chain complementarity determining regions, HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity determining regions, LCDR1, LCDR2, and LCDR3, wherein the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences shown in Table 1, and the corresponding set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences shown in Table 2.

[0011] In one embodiment, the anti-CD180 binding molecule comprises a heavy chain variable region and a light chain variable region having the sequences disclosed herein.

[0012] In one embodiment, the anti-CD180 binding molecule comprises heavy and light chains having the sequences disclosed herein.

[0013] In one embodiment, the anti-CD180 binding molecule comprises an IgG, Fv, scFv, Fab, F(ab'), minibody, diabody, triabody, nanobody, bispecific antibody, single-domain antibody, or chimeric antigen receptor. In one embodiment, the IgG is IgG1, IgG2, IgG3, or IgG4. In one embodiment, the bispecific anti-CD180 antibody is a biparatopic antibody, i.e., a bispecific antibody that targets two non-overlapping epitopes on the same target antigen. In some embodiments, the bispecific antibody binds to CD180 and CD123 ("CD180xCD123 bispecific antibody"). In one embodiment, the bispecific anti-CD180 antibody comprises a variable heavy chain (VH) region and a variable light chain (VL) region and / or CDRs disclosed herein that have binding specificity for CD180, and a combination of VH, VL, and / or CDRs disclosed herein, That is, it comprises a set of three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the set of HCDR1, HCDR2, and HCDR3 and the corresponding set of LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences disclosed herein. In another embodiment, the present disclosure provides an isolated polynucleotide sequence encoding the anti-CD180 binding molecule disclosed herein. In another embodiment, the present disclosure provides a vector comprising the polynucleotide sequence disclosed herein. In another embodiment, the present disclosure provides a host cell comprising the vector disclosed herein.

[0014] In one embodiment, the present disclosure provides a composition comprising a pharmaceutically acceptable carrier and any one of the anti-CD180 binding molecules disclosed herein. In one embodiment, the composition comprises an antibody-drug conjugate. In one embodiment, the antibody-drug conjugate comprises tesirin. In some embodiments, the antibody-drug conjugate comprises deruxtecan.

[0015] In another embodiment, the present disclosure provides a composition comprising any of the polynucleotide sequences disclosed herein. In one embodiment, the polynucleotide in such a composition comprises an expression vector for expressing an anti-CD180 binding molecule in a cell.

[0016] The present disclosure provides an anti-CD180 antibody drug conjugate (ADC) comprising a monoclonal antibody or antigen-binding fragment thereof that targets tumors that highly express CD180, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets tumors that highly express CD180 to the cytotoxic drug payload; The anti-CD180 antibody or antigen-binding fragment thereof comprises a set of three heavy chain complementarity-determining regions, HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity-determining regions, LCDR1, LCDR2, and LCDR3; The set of HCDR1, HCDR2 and HCDR3 and the corresponding set of LCDR1, LCDR2 and LCDR3 each comprise the following amino acid sequences: HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5 and 15; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 45; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 54 to 56 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57 to 59; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 68 to 70 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57, 58, and 71; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 80-82; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 91-93; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 102, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 103, and 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 112, 5, and 113; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 130; Or HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6.

[0017] The present disclosure also provides an anti-CD180 antibody-drug conjugate (ADC), comprising a monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors to the cytotoxic drug payload, wherein the anti-CD180 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and light chain variable region comprise the amino acid sequence of SEQ ID NO: 7-8, SEQ ID NO: 16-17, SEQ ID NO: 27-28, SEQ ID NO: 36-37, SEQ ID NO: 46-47, SEQ ID NO: 60-61, SEQ ID NO: 72-73, SEQ ID NO: 83-84, SEQ ID NO: 94-95, SEQ ID NO: 104-105, SEQ ID NO: 114-115, SEQ ID NO: 122-123, SEQ ID NO: 131-132, or SEQ ID NO: 139-140.

[0018] The present disclosure provides an anti-CD180 antibody-drug conjugate (ADC) comprising a monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors to the cytotoxic drug payload, wherein the anti-CD180 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, and the heavy chain and light chain comprise the amino acid sequence of SEQ ID NOs: 11-12, 20-21, 31-32, 40-41, 50-51, 64-65, 76-77, 87-88, 98-99, 108-109, 118-119, 126-127, 135-136, or 143-144.

[0019] In one particular embodiment of the provided ADC, the cytotoxic drug payload comprises the ADC drug-linker conjugate tesirin (SG3249), wherein tesirin comprises the cytotoxic drug payload; the cytotoxic drug payload comprises a pyrrolobenzodiazepine dimer cytotoxic DNA alkylating drug (SG3199), wherein tesirin is conjugated via a cleavable linker moiety to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-high expressing tumors.

[0020] In one particular embodiment of the provided ADC, the ADC comprises an ADC drug-linker conjugate deruxtecan, the deruxtecan comprising a cleavable linker, a self-immolative aminomethylene spacer, and a cytotoxic drug payload, the cytotoxic drug payload comprising a topoisomerase 1 inhibitor payload, the topoisomerase 1 inhibitor payload being a derivative of exatecan (DX-8951), and the deruxtecan is conjugated to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-high-expressing tumors via the cleavable linker. In one embodiment, the cleavable linker is a glycine-glycine-phenylalanine-glycine tetrapeptide-based linker.

[0021] In another embodiment, the present disclosure provides a method of modulating a CD180-mediated immune response in a subject, comprising administering to the subject a composition comprising any one of the anti-CD180 binding molecules disclosed herein. In one embodiment, the composition comprises an antibody-drug conjugate provided herein. In another embodiment, the present disclosure provides a method of modulating a CD180-mediated immune response in a subject, comprising administering to the subject a composition comprising any one of the polynucleotide sequences disclosed herein.

[0022] In another embodiment, the present disclosure provides a method of treating a disease in a subject, comprising administering to the subject a composition comprising any one of the anti-CD180 binding molecules disclosed herein. In one embodiment, the composition comprises an antibody drug conjugate. In one embodiment, the antibody drug conjugate comprises tesirin (an ADC drug linker conjugate). In a particular embodiment, the antibody drug conjugate comprises deruxtecan (an ADC drug linker conjugate). In some embodiments, the present disclosure provides a method of treating a disease in a subject, comprising administering to the subject a composition comprising any one of the polynucleotide sequences disclosed herein.

[0023] These and other aspects of the present invention will be understood from the following drawings and detailed description of the invention.

[0024] Several embodiments of the present invention are herein described, by way of example only, with reference to the accompanying drawings. It is emphasized that, with detailed reference now to the accompanying drawings, the specific matters shown are exemplary and are for purposes of illustrating embodiments of the invention. In this regard, the description taken in conjunction with the accompanying drawings will make apparent to those skilled in the art how embodiments of the invention may be practiced. [Brief explanation of the drawings]

[0025] [Figure 1A-C]Figure 1 shows the cell-binding characteristics of anti-CD180 antibodies. All CHAMP mAbs were subjected to binding assays by cell-based ELISA. Figure 1A: Antibodies were screened at 100 nM and 30 nM in a cell-based ELISA using HEK293 cells expressing CD180 / MD-1. All CHAMP mAbs showed significant binding by ELISA. P84, mouse IgG, and secondary-only mouse were negative controls. MAS-17729 and MHR3-11 were commercially available positive control antibodies. Figure 1B: Antibodies were screened at 100 nM in a cell-based ELISA using HEK293 cells expressing MD-1 only. None of the CHAMP mAbs showed significant binding by ELISA. P84, mouse IgG, secondary-only mouse, MAS-17729, and MHR3-11 were negative control antibodies. Figure 1C: Antibodies were screened at 100 nM in a cell-based ELISA using the parental HEK293 cell line. None of the CHAMP-mAbs showed significant binding to the cells. P84, mouse IgG, secondary only mouse, MAS-17729, and MHR3-11 were negative control antibodies. [Figure 2] Four CHAMP mAbs were subjected to EC50 evaluation by cell-based ELISA using HEK293 cells expressing human D180 / MD-1. MAS-17729 and MHR-73-11 were commercially available positive controls. [Figure 3A-C] The intrinsic binding affinity of the antibodies, as measured by surface plasmon resonance (SPR) assay, is shown. The antibodies were captured in a bivalent format, and soluble human CD180 from Sino Biologics (Figure 3A), soluble human CD180 from Creative Biomart (Figure 3B), or soluble rhesus CD180 (Figure 3C) was passed over the captured antibodies. Only ChampmAb-006 and ChampmAb-007 showed binding to the soluble protein, suggesting that the epitopes for all other Champ mAbs are masked when CD180 is expressed as a soluble protein. The K, k, and K are shown for each condition. [Figure 4]Figure 1 shows the results of differential scanning fluorimetry (DSF) and turbidity assays used to predict the thermal aggregation rate of antibodies. DSF and turbidity assays were performed on all antibodies. In all assays, the antibodies showed acceptable melting temperatures (Tm) above 65°C. [Figure 5] Figure 1 shows the results of an affinity capture self-interaction nanoparticle spectroscopy (AC-SINS) assay used to test the potential for antibodies to interact with themselves. All CHAMP antibodies showed favorable profiles with less than an 11 nM shift, suggesting a low propensity for self-interaction. The FDA-approved antibodies infliximab, pembrolizumab, and rituximab were used as standards in this assay. [Figure 6] Size exclusion chromatography results are shown. All CHAMP antibodies demonstrate a high level of purity (>95%) after production, both before and after a freeze / thaw cycle (1xF / T). [Figure 7] Results of capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), an analytical method for assessing protein purity, are shown. All CHAMP mAbs tested by this method demonstrate high levels of purity (>95%) after production. [Figure 8] 1 shows the results of a baculovirus particle ELISA, which tests the tendency of antibodies to interact nonspecifically with proteins. All CHAMP mAbs show a low tendency for nonspecific polyreactive binding. [Figure 9] 1 shows one embodiment of antibody conjugation to the linker payload Tecilin. [Figure 10A-B] This figure shows the ex vivo cytotoxicity of ADCs in primary acute myeloid leukemia. Ex vivo cell killing assays were performed using primary acute myeloid leukemia (AML) derived from leukapheresis transfusion. Primary cell cultures were established in a 96-well plate format with 54 test wells. Test agents were added for 120 hours of incubation before Cell Titer Glo was added to test cell viability. The half-maximal inhibitory concentration (IC50) was calculated and plotted against RNA expression levels (transcripts per million, TPM) quantified by RNAseq. [Figure 11] This figure shows the inhibition of ex vivo cytotoxicity of ADC in primary acute myeloid leukemia.Ex vivo cell killing assay was performed using primary AML derived from leukapheresis transfusion.Primary cell culture was established in a 96-well plate format with 54 test wells.MHR73-11 was added to the cells at a concentration of 300ng / mL for 10 minutes.Test drug was added at a concentration of 300ng / mL for 120 hours of incubation before Cell Titer Glo was added to test cell viability. [Figure 12] This figure shows the ex vivo cytotoxicity of ADCs in primary mantle cell lymphoma samples. Ex vivo cell killing assays were performed using primary mantle cell lymphoma (MCL) derived from leukapheresis or blood. Primary cell cultures were established in a 96-well plate format with 54 test wells. Test agents were added for 120 hours of incubation before Cell Titer Glo was added to test cell viability. The half-maximal inhibitory concentration (IC50) was calculated. [Figures 13A-D]An overview of CD180 targets is shown. Figure 13A shows that CD180 surface expression is enriched in primary AML samples. The distribution of protein expression in the CD180 proteomics of primary AML correlates with cell surface expression in primary AML, and CD180 is more elevated in primary AML than in normal immune cells. Therefore, cytogenetic abnormalities can be used as a biomarker for first-in-human (FIH) trials. Figure 13B shows CD180 expression in primary cultures of normal bone marrow (BM), suggesting an improved therapeutic index (TI) compared with CD133 ADCs. CD180 expression is lower than CD133 expression in normal hematopoietic stem cells (HSCs) and progenitor cells, with expression highest in mature B cells and plasmacytoid dendritic (pDC) cells. Figure 13C shows that CD180 is expressed on leukemic stem cells (LSCs) and progenitor cells in primary AML, which would eliminate measurable residual disease (MRD). Figure 13D shows that enhanced inflammatory signaling is observed in CD180-high expressing tumors. Inflammatory AML provides a survival niche for LSCs. Primary AML with enhanced CD180 expression also exhibits elevated genes involved in the inflammatory process. Pathways involved in the innate immune response and oxidative stress response are enhanced in primary AML with high CD180 expression. [Figure 14A-G] Characterization of anti-CD180 antibody leads CHAMPmAB-001 and CHAMPmAB-014 is shown; melting temperature, self-interaction, freeze-thaw stability, purity (Figures 14A-14F); comparison of CHAMPmAB-001 and CHAMPmAB-014 with isotype controls (ATX-P-84, and mIgG1), with secondary controls (hIgG1, hu2'Ab only, and mu2'Ab only), and with no staining (control sample) for binding to human CD180 / MD1 (positive) cells, MD1 (negative) cells, and parental HEK cells (negative) (Figure 14F); and half-maximal effective concentrations (EC50) of CHAMPmAB-001 (6 nM) and CHAMPmAB-014 (4 nM) against HEK293 CD180 / MD-1 cells (Figure 14G). [Figure 15A-C]Figure 15A shows the conjugation of the lead anti-CD180 antibody CHAMPmAB-001 to tesirine compared to the conjugation of isotype IgG1 to tesirine. Figure 15A shows a schematic diagram of one embodiment of an ADC of an anti-CD180 antibody conjugated to the linker payload tesirine (x2). Figure 15B shows a drug-to-antibody ratio (DAR) of 2.1, i.e., the average number of drugs conjugated to CHAMPmAB-001, an anti-CD180 antibody of the present disclosure, conjugated to the linker payload tesirine (x2) (CO-ADC-001). Figure 15C shows a DAR of 2.2 for isotype IgG1 conjugated to the linker payload tesirine (x2) (CO-ADC-003). [Figures 16A-C] Figure 16 shows the conjugation of the lead anti-CD180 antibody CHAMPmAB-004 to deruxtecan compared to the conjugation of isotype IgG1 to deruxtecan. Figure 16A shows a schematic diagram of one embodiment of an anti-CD180 antibody conjugated to the linker payload deruxtecan (x8). Figure 16B shows the DAR of 8 for CHAMPmAB-001, an anti-CD180 antibody of the disclosure, conjugated to the linker payload deruxtecan (x8). Figure 16C shows the DAR of 8.97 for isotype IgG1 conjugated to the linker payload deruxtecan (x8). [Figures 17A-D]Figure 17 shows a therapeutic summary of potent CD180-dependent cytotoxicity observed in primary tumors. Figure 17A shows that the tesirin conjugate is a potent ADC in CTG-2240 primary AML; CO-ADC-001 is a therapeutic anti-CD180 mAb conjugated with tesirin (DAR2). Figure 17B shows that the deruxtecan conjugate is also a potent ADC in CTG-2240 primary AML, comparable to MYLOTARG®. CO-ADC-004 is a therapeutic anti-CD180 mAb conjugated with deruxtecan (DAR8). Figure 17C shows that the cytotoxicity of CO-ADC-001 correlates with CD180 expression in primary AML. Figure 17D shows that the cytotoxicity results of primary AML cells suggest a high frequency of responders. [Figures 18A-C] Figure 18A shows the percentage viability of primary AML cells after the addition of CO-ADC-001 (300 ng / mL) without an initial CD180 blocking Ab, compared to the percentage viability of primary AML cells after the addition of CD180 ADC when 500 ng / mL of naked CD180 antibody was added before the addition of CD180 ADC; the naked antibody blocked binding of the ADC to CD180. Figure 18B shows the percentage viability of primary AML cells after the addition of CO-ADC-002 (300 ng / mL) without an initial CD180 blocking Ab, compared to the percentage viability of primary AML cells after the addition of CD180 ADC when naked CD180 antibody (500 ng / mL), which inhibited binding of the ADC to CD180, was added before the addition of CD180 ADC. Figure 18C shows the percent viability of primary AML cells after addition of isotype ADC (300 ng / mL) without an initial CD180 blocking Ab compared to the percent viability of primary AML cells after addition of isotype ADC when naked CD180 antibody (500 ng / mL), which inhibited binding of the isotype ADC to CD180, was added prior to addition of the CD180 ADC. [Figure 19A-C]This shows that CD180 ADCs are potent against primary MCL. Figure 19A shows the percentage viability of primary MCL cells treated with CO-ADC-001, a therapeutic anti-CD180 mAb conjugated to Tecilin, compared with the percentage viability of primary MCL cells treated with CO-ADC-003, a negative control mAb conjugated to Tecilin. Figure 19A shows the percentage viability of primary MCL CTG-3446 treated with CO-ADC-001 and CO-ADC-003, respectively. Figure 19B shows the percentage viability of primary MCL CTG-3785 treated with CO-ADC-001 and CO-ADC-003, respectively. Figure 19C shows the percentage viability of primary MCL CTG-3448 treated with CO-ADC-001 and CO-ADC-003, respectively. [Figure 20A-B] Figure 20 shows that CO-ADC-001, a DAR2 Tessilin conjugate targeting CD180, is highly active against primary AML cells disseminated in vivo. Figure 20A shows the characteristics of primary AML CTG-2240. Figure 20B shows the results of in vivo analysis of tumor (%) in bone marrow, LSC in bone marrow, and CD123 cells in bone marrow after administration of 0.3MPK CO-ADC-001 compared to the control 0.3MPK and 0.3MPK IgG-ADC. [Figure 21A-C]Figure 21A shows the profile of primary AML CTG-2240 cells, a DAR8 deruxtecan conjugate targeting CD180. Figure 21B shows the results of in vivo analysis of the percentage of hCD45 tumors in the bone marrow, monocytes in the bone marrow, CD123 cells in the bone marrow, and CD180+ LSCs in the bone marrow, CD180+ monocytes in the bone marrow, CD180+CD117+ in the bone marrow, and CD180+CD123+ in the bone marrow after administration of 5MPK CO-ADC-004 compared to the control (vehicle) and 5MPK IgG-ADC (CO-ADC-005), as well as exemplary embodiments of the present invention, IgG-ADC or CO-ADC-004. FIG. 21C shows the results of in vivo analysis of CD180+ LSCs (%) in bone marrow, CD180+ monocytes (%) in bone marrow, CD180+CD117+ in bone marrow, and CD180+CD123+ in bone marrow. [Figures 22A-G]

[0023] Figure 1 shows the characteristics of one embodiment of the present invention, the lead anti-CD180 antibody CO-mAb-020 (CO-ADC-004), a fully human IgG1, including melting temperature, self-interaction, freeze-thaw stability, purity, polyreactivity, binding to CD180 / MD1 HEK293 cells, and internalization of CO-mAb-020 in MV-4-11, a human AML cell line established from the blast cells of a 10-year-old boy with mixed B-myelomonocytic leukemia (AML FAB M5) harboring the t(4;11) translocation and FLT3-ITD mutation. [Figure 23A-H]Figure 23A shows the conjugation of CO-mAB-20 to deruxtecan and tesirin. Figure 23A shows the anti-CD180 antibody lead CO-mAB-20, an embodiment of the present invention, conjugated to deruxtecan (Dxd) ×8, which has the chemical structure Dxd. Figure 23B shows a DAR of 8 for the conjugate of antibody CO-mAB-20 to deruxtecan (CO-ADC-004). Figure 23C shows a DAR of 8.97 for isotype IgG1 (CO-ADC-005) conjugated to the linker payload deruxtecan (×8). Figure 23D shows antibody CO-mAB-20 conjugated to tesirin ×2. Figure 23E shows a DAR of 2.1 for the conjugate of CO-mAB-20 to tesirin ×2 (CO-ADC-001). Figure 23F shows a DAR of 2.2 for the isotype IgG1 conjugate (CO-ADC-003) conjugated to the linker payload tesirin x2. Figures 23G-23H show that cell binding to HEK293 CD180 / MD-1 cells is unchanged after conjugation of CO-mAb-020 (CO-ADC-004) DAR:8 to deruxtecan (Figure 23G) compared to conjugation of CO-mAb-020 (CO-ADC-001) DAR:2.1 to tesirin (Figure 23H). The ADCs were subjected to half-maximal effective concentration (EC50) evaluation by cell-based ELISA using HEK293 cells expressing human CD180 / MD-1. [Figure 24A-B]These results demonstrate that CO-ADC-004, the lead ADC, is potent against MV4-11 tumors in vivo. CO-ADC-004 is a CD180-Dxd ADC. CO-ADC-005 is an isotype-Dxd ADC. Tail vein injection (TVI) of MV4-11 Luc was used for systemic engraftment. Mice were randomized once bioluminescence was detected above background in more than 90% of mice. A single dose of 5 mg / kg (MPK) was administered to each mouse via intravenous injection of the therapeutic agent (either CO-ADC-004 at 5MPK or CO-ADC-005 at 5MPK). Tumor burden was assessed by bioluminescence (Figure 24B). CO-ADC-004 administered at a dose of 5MPK demonstrated complete regression for over 30 days compared to the 5MPK isotype-Dxd ADC (CO-ADC-005) (Figure 24A). [Figure 25A-B] These results demonstrate that CO-ADC-001 ADC is potent against MV4-11 tumors in vivo. CO-ADC-001 is a CD180-Tesirin ADC. CO-ADC-003 is an isotype-Tesirin ADC. MV4-11 Luc tail vein injection (TVI) was used for systemic engraftment. Mice were randomized once bioluminescence was detected above background in more than 90% of mice. A single dose of 5 mg / kg (MPK) was administered to each mouse via intravenous injection of the therapeutic agent (either CO-ADC-001 ADC or CO-ADC-003). Tumor burden was assessed by bioluminescence (Figure 25B). CO-ADC-001 ADC administered at a dose of 15 MPK demonstrated complete regression for more than 30 days compared to the isotype-Tesirin ADC CO-ADC-003 (Figure 25A). [Figure 26A-C] Figures 26A-26B show that CD180 protein expression correlates with ADC cytotoxicity. Figures 26A-26B show a wide range of sensitivity and cytotoxicity in AML patient samples. Figure 26C shows the correlation between CD180 protein expression and IC50. [Figure 27A-C]Correlation between receptor expression and ADC efficacy for other ADCs is shown. Figure 27A shows that CD19 expression correlates with the in vitro activity of roncatecillin (loncatuximabtesillin-LPYL). Figure 27B shows that CD33 expression correlates with the in vitro activity of IMGN779, an anti-CD33 ADC with a DNA-alkylated IGN (indolinobenzodiazepine pseudodimer) payload and a cleavable s-SPDB linker. Figure 27C shows that CD123 expression correlates with the in vitro activity of SGN-CD123A, an antibody-drug conjugate consisting of a humanized CD123 antibody conjugated to a pyrrolobenzodiazepine dimer (PBD) via a stable, protease-cleavable dipeptide linker, with engineered cysteines on each heavy chain attaching the PBD to the antibody for site-specific conjugation. DETAILED DESCRIPTION OF THE INVENTION

[0026] Unless the context clearly indicates otherwise, throughout this specification and the claims, words such as "comprise," "comprising," and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; i.e., meaning "including," but not limited to. Terms using the singular or plural also include the plural or singular, respectively. Furthermore, words of similar meaning, such as "herein," "above," and "below," when used in this application, shall refer to this application as a whole and not to any particular portions of this application. The singular forms "a," "an," and "the" herein include plural referents unless the context clearly dictates otherwise. As used herein, "and" is used synonymously with "or," unless expressly indicated otherwise.

[0027] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention, exemplary methods and / or materials are described below. In the event of a conflict, the present patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are intended to be illustrative only and are not necessarily limiting. Each literature reference or citation cited herein is incorporated herein by reference in its entirety.

[0028] In the description herein, each of the steps of the present invention and variations thereof are described. It is to be understood that the description herein is not intended to be limiting, and that changes in the components, sequence of steps, and other variations are also within the scope of the present invention.

[0029] It should be understood that, for clarity, certain features of the invention that are described in separate embodiments may also be demonstrated in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment may also be demonstrated individually, or in any suitable subcombination, or as preferred in any other embodiment described in the invention. Certain features described in the context of various embodiments are not considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0030] Throughout this application, various embodiments of the present disclosure may be expressed in a range format. It should be understood that the description in range format is merely for convenience or brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be construed as including all specifically disclosed subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be construed as including specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This is true regardless of the breadth of the range.

[0031] Whenever a range of values ​​is given herein, it is intended to include any recited number (fractional or integer) within the given range. The phrases "ranging between" a first denoted number and a second denoted number, and "ranging from" a first denoted number to a second denoted number, are used synonymously herein and are intended to include the first and second denoted numbers and all fractional and integer values ​​therebetween.

[0032] When values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable. In one embodiment, the term "about" refers to a 0.1 to 5% variation of the stated number or numerical range. In another embodiment, the term "about" refers to a 1 to 10% variation of the stated number or numerical range. In another embodiment, the term "about" refers to up to a 20% variation from the stated number or numerical range. In one embodiment, the term "about" refers to a ±10% variation of the stated number or numerical range. In another embodiment, the term "about" refers to a ±5% variation of the stated number or numerical range.

[0033] In one embodiment, the present disclosure describes the isolation and use of a panel of anti-CD180 binding molecules. In one embodiment, the anti-CD180 binding molecules comprise a set of three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 as disclosed herein and a corresponding set of three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 as disclosed herein.

[0034] In one embodiment, the anti-CD180 binding molecule comprises an anti-CD180 antibody. The term "antibody" herein may be used synonymously with the term "immunoglobulin," with all the same properties and meanings. The antibody binding domain or antigen-binding site may be a fragment of an antibody or a genetically engineered product of one or more fragments of an antibody, which fragment is responsible for specific binding to a target antigen. "Specifically binds" means that the binding is selective for the antigen of interest and can be distinguished from unwanted or non-specific interactions. For example, an antibody may have an equilibrium dissociation constant of ≦10 -5 , 10 -6 , or 10 -7 M. In some embodiments, the equilibrium dissociation constant is ≦10 -8 M or 10 -9 M. In some further embodiments, the equilibrium dissociation constant is ≦10 -10 M, 10 -11 M, or 10 -12 M. In some embodiments, the equilibrium dissociation constant is ≦10 -5 M~10 -12 M may be in the range.

[0035] An "epitope" or "antigenic determinant" refers to a site on an antigen to which an antibody binds. Epitopes can be formed both by contiguous amino acids or by noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically comprises at least three amino acids, more usually at least five or eight to ten amino acids, in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, "Epitope Mapping Protocols" in Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996).

[0036] As used herein, the term "antibody" encompasses antibody fragments or fragments that retain binding specificity, including, but not limited to, IgG, heavy chain variable regions (VH), light chain variable regions (VL), Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, nanobodies, minibodies, diabodies, triabodies, tetrabodies, and single domain antibodies (see, e.g., Hudson and Souriau, Nature Med. 9:129-134 (2003)). Also included are humanized antibodies, primatized antibodies, and chimeric antibodies, as these terms are commonly understood in the art.

[0037] As used herein, the term "heavy chain variable region" may be used synonymously with the term "VH domain" or "VH," with all the same meanings and properties. As used herein, the term "light chain variable region" may be used synonymously with the term "VL domain" or "VL," with all the same meanings and properties. Those skilled in the art will understand that a "heavy chain variable region" or "VH" with respect to an antibody encompasses a fragment of a heavy chain comprising three complementarity-determining regions (CDRs) interposed between adjacent sections known as framework regions. Framework regions are more highly conserved than CDRs and form a framework supporting the CDRs. Similarly, those skilled in the art will also understand that a "light chain variable region" or "VL" with respect to an antibody encompasses a fragment of a light chain comprising three CDRs interposed between framework regions.

[0038] As used herein, the term "complementarity-determining region" or "CDR" refers to the hypervariable region of a heavy or light chain variable region. Starting from the N-terminus, each heavy or light chain polypeptide has three CDRs designated "CDR1," "CDR2," and "CDR3." Crystallographic analysis of numerous antigen / antibody conjugates has shown that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Thus, the CDR regions are primarily responsible for the specificity of the antigen-binding site. In one embodiment, the antigen-binding site comprises six CDRs, including CDRs from each of the heavy and light chain variable regions.

[0039] Those skilled in the art will appreciate that an scFv is a fusion polypeptide comprising the variable heavy (VH) and variable light (VL) regions of an immunoglobulin linked by a short linker peptide, which may have, for example, from 10 to about 25 amino acids.

[0040] Those skilled in the art will also understand that the term "Fab," with respect to an antibody, generally includes a portion of an antibody consisting of a single light chain (both variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain, while F(ab')2 includes a fragment of a heavy chain containing a VH domain and a light chain containing a VL domain.

[0041] In some embodiments, antibodies include monoclonal and polyclonal antibodies, including whole antibody molecules. In some embodiments, antibodies include antibody fragments or multiple fragments that retain binding specificity, including, but not limited to, variable heavy (VH) fragments, variable light (VL) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.

[0042] In some embodiments, the anti-CD180 binding molecules of the present disclosure may be chimeric antibodies. A "chimeric antibody" is an immunoglobulin molecule in which the constant region or a portion thereof has been modified, substituted, or exchanged, such that the antigen-binding site (variable region) is linked to a constant region of a different or modified class, effector function, and / or species, or to an entirely different molecule, such as an enzyme, toxin, hormone, growth factor, drug, etc., that confers novel properties to the chimeric antibody.

[0043] In some embodiments, the anti-CD180 binding molecules of the present disclosure may be humanized antibodies. A "humanized antibody" is an immunoglobulin molecule that contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody). In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR and framework sequences. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are of human immunoglobulin consensus sequence. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)). Humanization can be performed essentially according to the method of Winter and coworkers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by replacing the sequence of a rodent CDR or CDRs with the corresponding sequence of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.

[0044] In some embodiments, the anti-CD180 binding molecules of the present disclosure are bispecific (or multispecific) antibodies. As generally known in the art, bispecific antibodies are recombinant proteins comprising antigen-binding fragments of two different monoclonal antibodies, thereby capable of binding to two different antigens. In some embodiments, bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificity for at least one other antigen in addition to CD180, or for different CD180 epitopes. Similarly, multispecific antibodies are recombinant proteins comprising antigen-binding fragments of at least two different monoclonal antibodies (e.g., two, three, or four different monoclonal antibodies). In one embodiment, the bispecific anti-CD180 antibody is a biparatopic antibody, i.e., a bispecific antibody that targets two non-overlapping epitopes on the same target antigen. In a specific embodiment, the bispecific antibody binds to CD180 and CD123 (a "CD180xCD123 bispecific antibody"). In some embodiments, the bispecific anti-CD180 antibody comprises a combination of variable heavy chain (VH) and variable light chain (VL) regions and / or CDRs disclosed herein, and variable heavy chain (VH) and variable light chain (VL) regions and / or CDRs disclosed herein, that have binding specificity for CD180, i.e., a set of three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the set of HCDR1, HCDR2, and HCDR3 and the corresponding set of LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences disclosed herein.

[0045] In another embodiment, one skilled in the art can readily use the VH, VL, and / or CDRs disclosed herein to construct a chimeric antigen receptor (CAR) with binding specificity for CD180.

[0046] In one embodiment, the anti-CD180 antibody or antigen-binding fragment thereof may comprise one or more Fc domain mutations that weaken binding to an FcγR receptor (e.g., FcγI, FcγIIa, FcγIIb, or FcγRIIIa). Any suitable Fc domain mutant can be used, which reduces binding of the Fc domain to an FcγR receptor, e.g., reduces binding by at least 50% compared to binding to the non-mutated Fc domain. Fc mutations and truncations that can reduce binding to an FcγR receptor can be made by one of skill in the art based on techniques well known in the art.

[0047] In some embodiments, the anti-CD180 binding molecules or anti-CD180 antibodies of the present disclosure can be further conjugated to an effector moiety. The effector moiety can be any number of molecules, including a labeling moiety, such as a radioactive or fluorescent label, or a therapeutic moiety. When the effector moiety is a therapeutic moiety, it is typically a cytotoxic agent. Cytotoxic agents are numerous and varied, including, but not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments are well known in the art. Cytotoxic agents also include radioactive chemicals created by conjugating a radioisotope to an antibody or by binding a radionuclide to a chelator linked to the antibody.

[0048] In another embodiment, the anti-CD180 binding molecule or anti-CD180 antibody of the present disclosure may be modified to extend its half-life, such as by attaching at least one molecule to the antibody to extend its serum half-life, including, but not limited to, a polyethylene glycol (PEG) group, serum albumin, transferrin, a transferrin receptor or a transferrin-binding portion of the receptor, or a combination thereof. As used herein, the term "linked" refers to a covalently or non-covalently conjugated substance. Conjugation may be by genetic engineering or chemical means.

[0049] Anti-CD180 binding molecule The present disclosure provides numerous anti-CD180 binding molecules comprising a set of three heavy chain complementarity determining regions, HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity determining regions, LCDR1, LCDR2, and LCDR3, wherein the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences shown in Table 1, and the corresponding set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences shown in Table 2. For convenience, taking Ab-001 as an example (see Table 1), the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs: 1 to 3, respectively (see Table 1), while the corresponding set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs: 4 to 6, respectively (see Table 2).

[0050] In one embodiment, a set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively, and a corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 4, 5 and 15, respectively. In one embodiment, a set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 1, 24 and 25, respectively, and a corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 4, 5 and 26, respectively. In one embodiment, a set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 1, 35 and 25, respectively, and a corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 4, 5 and 26, respectively. In one embodiment, a set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 1, 44 and 25, respectively, and a corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 4, 5 and 45, respectively. In one embodiment, a set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 54 to 56, respectively, and a corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 57 to 59, respectively. In one embodiment, a set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 68 to 70, respectively, and a corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 57, 58 and 71, respectively. In one embodiment, a set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 1, 24 and 25, respectively, and a corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 80 to 82, respectively. In one embodiment, a set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 1, 35 and 25, respectively, and a corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 91 to 93, respectively.In one embodiment, the set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 1, 102, 25, respectively, and the corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 4, 103, 6, respectively. In one embodiment, the set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 1, 24, 25, respectively, and the corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 112, 5, 113, respectively. In one embodiment, the set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 1, 35, 25, respectively, and the corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 4, 5, 6, respectively. In one embodiment, the set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 1, 44, 25, respectively, and the corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 4, 5, 130, respectively. In one embodiment, the set of HCDR1, HCDR2 and HCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 1, 24, 25, respectively, and the corresponding set of LCDR1, LCDR2 and LCDR3 comprises the amino acid sequences set forth in SEQ ID NOs: 4, 5, 6, respectively.

[0051] In another embodiment, the anti-CD180 binding molecules disclosed herein comprise heavy and light chain CDR sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above, for example, but not limited to, identity as determined by the National Center for Biotechnology Information (NCBI) BlastP software using default parameters.

[0052] The term "identity," when used in reference to polypeptide (or nucleotide) sequences, refers to the degree of identity between two or more polypeptide (or nucleotide) sequences or fragments thereof. Typically, the degree of similarity between two or more polypeptide (or nucleotide) sequences refers to the degree of similarity in terms of the composition, order, or arrangement of two or more amino acids or nucleotides of the two or more polypeptide (or nucleotide) sequences. Those skilled in the art will understand that percent identity (% identity) is a number that indicates how similar a query sequence is to a target sequence. The higher the percent identity, the greater the match.

[0053] In another embodiment, an anti-CD180 binding molecule disclosed herein comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region comprise the amino acid sequence of SEQ ID NOs: 7 to 8, SEQ ID NOs: 16 to 17, SEQ ID NOs: 27 to 28, SEQ ID NOs: 36 to 37, SEQ ID NOs: 46 to 47, SEQ ID NOs: 60 to 61, SEQ ID NOs: 72 to 73, SEQ ID NOs: 83 to 84, SEQ ID NOs: 94 to 95, SEQ ID NOs: 104 to 105, SEQ ID NOs: 114 to 115, SEQ ID NOs: 122 to 123, SEQ ID NOs: 131 to 132, or SEQ ID NOs: 139 to 140.

[0054] In another embodiment, the anti-CD180 binding molecules disclosed herein comprise heavy and light chain variable regions that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above, for example, but not limited to, identity as determined by the National Center for Biotechnology Information (NCBI) BlastP software using default parameters.

[0055] In another embodiment, an anti-CD180 binding molecule disclosed herein comprises a heavy chain and a light chain, wherein the heavy chain and the light chain comprise the amino acid sequence of SEQ ID NOs: 11-12, 20-21, 31-32, 40-41, 50-51, 64-65, 76-77, 87-88, 98-99, 108-109, 118-119, 126-127, 135-136, or 143-144.

[0056] In another embodiment, the anti-CD180 binding molecules disclosed herein comprise heavy and light chains that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above, for example, but not limited to, identity as determined by the National Center for Biotechnology Information (NCBI) BlastP software using default parameters.

[0057] In another embodiment, an isolated polynucleotide sequence encoding an anti-CD180 binding molecule disclosed herein is provided. In one embodiment, the anti-CD180 binding molecule comprises a heavy chain variable region encoded by a nucleotide sequence having the sequence of SEQ ID NO: 9, 18, 29, 38, 48, 62, 74, 85, 96, 106, 116, 124, 133, or 141. In one embodiment, the anti-CD180 binding molecule comprises a light chain variable region encoded by a nucleotide sequence having the sequence of SEQ ID NO: 10, 19, 30, 39, 49, 63, 75, 86, 97, 107, 117, 125, 134, or 142. In one embodiment, the anti-CD180 binding molecule comprises a heavy chain encoded by a nucleotide sequence having the sequence of SEQ ID NO: 13, 22, 33, 42, 52, 66, 78, 89, 100, 110, 120, 128, 137, or 145. In one embodiment, the anti-CD180 binding molecule comprises a light chain encoded by a nucleotide sequence having the sequence of SEQ ID NO: 14, 23, 34, 43, 53, 67, 79, 90, 101, 111, 121, 129, 138 or 146.

[0058] In another embodiment, the present disclosure includes polynucleotide sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the above-described polynucleotide sequences, as determined by methods and parameters generally known in the art.

[0059] Composition used In one embodiment, the present disclosure also provides a composition comprising an anti-CD180 binding molecule disclosed herein and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers are well known in the art. For example, "Remington's Pharmaceutical Science" by E.W. Martin (Mack Publishing Co., Easton, PA, 23rd Edition, 2020) describes compositions and formulations suitable for pharmaceutical delivery of the polypeptides or antibodies disclosed herein. In one embodiment, the composition comprises an antibody-drug conjugate disclosed herein. In one embodiment, the antibody-drug conjugate comprises tesirin. In some embodiments, the ADC comprises deruxtecan.

[0060] Those skilled in the art can readily incorporate the anti-CD180 binding molecules disclosed herein into therapeutic agents that target cells expressing CD180. Examples of such therapeutic modalities include, but are not limited to, monoclonal antibodies, antibody-drug conjugates, chimeric antigen receptor T cells, and chimeric antigen receptor natural killer cells. In one embodiment, a therapeutic agent can be used to stimulate or activate CD180-expressing cells.

[0061] Compositions comprising the anti-CD180 binding molecules or antigen-binding fragments thereof disclosed herein can be administered to a subject (e.g., a human or animal) alone or in combination with a carrier, i.e., a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable; i.e., the material can be administered to a subject without causing any undesired biological effects or adversely interacting with any other components of the pharmaceutical composition in which it is contained. As is well known to those skilled in the art, a carrier will be selected to minimize degradation of the polypeptides disclosed herein and to minimize adverse side effects in the subject. Pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical arts.

[0062] In one embodiment, the composition comprises an anti-CD180 binding molecule comprising a set of three heavy chain complementarity determining regions, HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity determining regions, LCDR1, LCDR2, and LCDR3, wherein the set of HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences shown in Table 1, and the corresponding set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences shown in Table 2.

[0063] In another embodiment, the present disclosure also provides a composition comprising any of the polynucleotide sequences disclosed herein, wherein the polynucleotide sequence encodes an anti-CD180 binding molecule disclosed herein.

[0064] Compositions comprising the anti-CD180 binding molecules or antigen-binding fragments thereof disclosed herein, or the polynucleotide sequences disclosed herein encoding the anti-CD180 binding molecules, can be administered (e.g., to a mammal, cell, or tissue) in any suitable manner, depending on whether local or systemic treatment is desired. For example, compositions can be administered topically (e.g., ophthalmically, intravaginally, rectally, intranasally, transdermally, etc.), orally, by inhalation, or parenterally (including by intravenous drip or subcutaneous, intracavitary, intraperitoneal, intradermal, or intramuscular injection). Local intranasal administration refers to delivery of a composition into the nose or intranasal cavity via one or both nostrils. Compositions can be delivered by a spray or droplet mechanism, or by aerosolization. Alternatively, administration can be intratumoral, e.g., by local or intravenous injection.

[0065] When the composition is administered parenterally, administration is generally by injection.Injectable preparations can be prepared in conventional form, either as a solution or suspension, as a solid form suitable for suspension in liquid before injection, or as emulsion.In addition, parenteral administration can include the preparation of a sustained release system or a sustained release system to maintain a constant dosage.

[0066] The present disclosure provides an anti-CD180 antibody drug conjugate (ADC) comprising a monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors to the cytotoxic drug payload, wherein the anti-CD180 antibody or antigen-binding fragment thereof comprises a set of three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and the set of HCDR1, HCDR2, and HCDR3 and the corresponding set of LCDR1, LCDR2, and LCDR3 each comprise the following amino acid sequences: HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5 and 15; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 45; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 54 to 56 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57 to 59; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 68 to 70 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57, 58, and 71; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 80-82; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 91-93; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 102, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 103, and 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 112, 5, and 113; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 130; or HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6.

[0067] The present disclosure provides an anti-CD180 antibody-drug conjugate (ADC), comprising a monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors to the cytotoxic drug payload, wherein the anti-CD180 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and light chain variable region comprise the amino acid sequence of SEQ ID NOs: 7-8, SEQ ID NOs: 16-17, SEQ ID NOs: 27-28, SEQ ID NOs: 36-37, SEQ ID NOs: 46-47, SEQ ID NOs: 60-61, SEQ ID NOs: 72-73, SEQ ID NOs: 83-84, SEQ ID NOs: 94-95, SEQ ID NOs: 104-105, SEQ ID NOs: 114-115, SEQ ID NOs: 122-123, SEQ ID NOs: 131-132, or SEQ ID NOs: 139-140.

[0068] The present disclosure provides an anti-CD180 antibody-drug conjugate (ADC) comprising a monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors to the cytotoxic drug payload, wherein the anti-CD180 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, and the heavy chain and light chain comprise the amino acid sequence of SEQ ID NOs: 11-12, SEQ ID NOs: 20-21, SEQ ID NOs: 31-32, SEQ ID NOs: 40-41, SEQ ID NOs: 50-51, SEQ ID NOs: 64-65, SEQ ID NOs: 76-77, SEQ ID NOs: 87-88, SEQ ID NOs: 98-99, SEQ ID NOs: 108-109, SEQ ID NOs: 118-119, SEQ ID NOs: 126-127, SEQ ID NOs: 135-136, or SEQ ID NOs: 143-144.

[0069] In one embodiment of the ADCs provided herein, the anti-CD180 antibody or antigen-binding fragment thereof comprises an IgG, Fv, scFv, Fab, F(ab')2, minibody, diabody, triabody, nanobody, bispecific antibody, single-domain antibody, or chimeric antigen receptor. In one embodiment, the bispecific anti-CD180 antibody is a biparatopic antibody, i.e., a bispecific antibody that targets two non-overlapping epitopes on the same target antigen. In a particular embodiment, the bispecific antibody binds to CD180 and CD123 (a "CD180xCD123 bispecific antibody"). In some embodiments, the bispecific anti-CD180 antibody comprises a variable heavy chain (VH) region and a variable light chain (VL) region and / or CDRs disclosed herein and a combination of VH, VL, and / or CDRs disclosed herein, i.e., a set of three heavy chain complementarity determining regions, HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity determining regions, LCDR1, LCDR2, and LCDR3, which have binding specificity for CD180, and each of the set of HCDR1, HCDR2, and HCDR3 and the corresponding set of LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences disclosed herein.

[0070] In some embodiments of the ADC, the IgG is IgG1, IgG2, IgG3, or IgG4. In one particular embodiment of the ADC, the cytotoxic agent payload comprises the ADC drug-linker conjugate Tesirine (SG3249), wherein Tesirine comprises the cytotoxic agent payload, wherein the cytotoxic agent payload comprises a pyrrolobenzodiazepine dimer cytotoxic DNA alkylating agent (SG3199), and Tesirine is conjugated to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-high-expressing tumors via a cleavable linker moiety.

[0071] In some embodiments of the ADC, the ADC comprises an ADC drug-linker conjugate deruxtecan, wherein the deruxtecan comprises a cleavable linker, a self-immolative aminomethylene spacer, and a cytotoxic drug payload, wherein the cytotoxic drug payload comprises a topoisomerase 1 inhibitor payload, wherein the topoisomerase 1 inhibitor payload is a derivative of exatecan (DX-8951), and wherein the deruxetan is conjugated to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-high-expressing tumors via a cleavable linker, wherein the cleavable linker is a glycine-glycine-phenylalanine-glycine tetrapeptide-based linker.

[0072] In certain embodiments, the cleavable linker moiety is a hydrazone linker, a disulfide linker, or a peptide linker. In various embodiments of the ADCs, the peptide linker is a dipeptide linker selected from the group consisting of valine-citrulline (Val-Cit), valine-alanine (Val-Ala), and alanine-alanine (Ala-Ala). In one embodiment, the dipeptide linker is linked to the cytotoxic drug payload by a spacer unit, and the spacer unit is para-aminobenzyloxycarbonyl (PABC). In some embodiments of the ADCs provided herein, the peptide linker is a tripeptide linker, and the tripeptide linker is a glutamic acid-valine-citrulline (EVCit) tripeptide linker. In one embodiment, the glutamic acid-valine-citrulline (EVCit) tripeptide linker is linked to a meta-amido para-aminobenzylcarbamate (MA-PABC) group. In one particular embodiment of the described and provided ADCs comprising the ADC drug-linker conjugate tesirin (SG3249), the drug-to-antibody ratio (DAR) is 2.1. In some embodiments of the ADCs comprising the ADC drug-linker conjugate deruxtecan, the drug-to-antibody ratio (DAR) is 8.

[0073] The present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an ADC comprising a monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors to the cytotoxic drug payload, wherein the monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors is an anti-CD180 antibody or antigen-binding fragment thereof that comprises a set of three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, and wherein the set of HCDR1, HCDR2, and HCDR3 and the corresponding set of LCDR1, LCDR2, and LCDR3 each comprise the following amino acid sequences: HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5 and 15; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 45; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 54 to 56 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57 to 59; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 68 to 70 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57, 58, and 71; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 80-82; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 91-93; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 102, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 103, and 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 112, 5, and 113; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 130; or HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6.

[0074] The present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an ADC comprising a monoclonal antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor to the cytotoxic drug payload, wherein the monoclonal antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor comprises a heavy chain variable region and a light chain variable region. An anti-CD180 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequence of SEQ ID NOs: 7 to 8, 16 to 17, 27 to 28, 36 to 37, 46 to 47, 60 to 61, 72 to 73, 83 to 84, 94 to 95, 104 to 105, 114 to 115, 122 to 123, 131 to 132, or 139 to 140.

[0075] The present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an ADC comprising a monoclonal antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor to the cytotoxic drug payload, wherein the monoclonal antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor is an antibody comprising a heavy chain and a light chain. A CD180 antibody or an antigen-binding fragment thereof, wherein the heavy chain and light chain comprise the amino acid sequence of SEQ ID NOs: 11 to 12, 20 to 21, 31 to 32, 40 to 41, 50 to 51, 64 to 65, 76 to 77, 87 to 88, 98 to 99, 108 to 109, 118 to 119, 126 to 127, 135 to 136, or 143 to 144.

[0076] The present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an ADC: The ADC comprises a monoclonal antibody or antigen-binding fragment thereof that targets a CD180-high expressing tumor, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets a CD180-high expressing tumor to the cytotoxic drug payload, wherein the cytotoxic drug payload comprises the ADC drug-linker conjugate tesirin (SG3249), wherein the tesirin comprises the cytotoxic drug payload, wherein the cytotoxic drug payload comprises a pyrrolobenzodiazepine dimer cytotoxic DNA alkylating drug (SG3199), and the tesirin is linked to the ADC via a cleavable linker moiety. and conjugated to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors, wherein the monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors is an anti-CD180 antibody or antigen-binding fragment thereof comprising a set of three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 and a corresponding set of three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein the set of HCDR1, HCDR2, and HCDR3 and the corresponding set of LCDR1, LCDR2, and LCDR3 each comprise the following amino acid sequences: HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5 and 15; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 45; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 54 to 56 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57 to 59; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 68 to 70 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57, 58, and 71; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 80-82; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 91-93; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 102, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 103, and 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 112, 5, and 113; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 130; or HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6.

[0077] The present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an ADC comprising a monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors, a cytotoxic drug payload, and a linker moiety conjugating the antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors to the cytotoxic drug payload, wherein the ADC comprises the ADC drug-linker conjugate deruxetan, wherein the deruxetan comprises a cleavable linker, a self-immolative aminomethylene spacer, and a cytotoxic drug payload, wherein the cytotoxic drug payload comprises a topoisomerase 1 inhibitor payload, wherein the topoisomerase 1 inhibitor payload is a derivative of exatecan (DX-8951), and the deruxetan is conjugated to the monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors via the cleavable linker, wherein the cleavable linker is a glycine-glycine-phenylalanine-glycine tetrapeptide-based linker.

[0078] In some embodiments, the cancer is acute myeloid leukemia (AML), mantle cell lymphoma (MCL), multiple myeloma, follicular lymphoma, acute B-lymphoblastic leukemia, or diffuse large B-cell lymphoma. In certain embodiments, the AML is primary AML or primary MCL. In one embodiment, the ADC comprises a set of HCDR1, HCDR2, and HCDR3 comprising the amino acids of SEQ ID NOs: 1-3, and a corresponding set of LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 4-6, and the cytotoxic drug payload comprises the pyrrolobenzodiazepine dimer cytotoxic alkylating agent Tesirine (SG3199) conjugated to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-high-expressing tumors via a cleavable linker moiety, wherein the cleavable linker moiety is a valine-alanine (Val-Ala) peptide linker. In one particular embodiment, the Val-Ala peptide linker is cleavable by cathepsin B. In one particular embodiment of the described and provided ADCs, comprising the ADC drug-linker conjugate Tecilin (SG3249), the drug-to-antibody ratio (DAR) is 2.1.

[0079] In one embodiment of the methods of treatment provided herein, the cancer is primary AML. In some embodiments of the methods provided, the cancer is primary MCL. In one specific embodiment, the ADC comprises a set of HCDR1, HCDR2 and HCDR3 comprising the amino acids of SEQ ID NOs: 1, 35 and 25, and a corresponding set of LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5 and 26, the ADC is conjugated to an ADC drug-linker conjugate deruxtecan, the deruxtecan comprising a glycine-glycine-phenylalanine-glycine tetrapeptide-based cleavable linker, a self-immolative aminomethylene spacer, and a cytotoxic drug payload, the cytotoxic drug payload comprising a topoisomerase 1 inhibitor payload, the topoisomerase 1 inhibitor payload is a derivative of exatecan (DX-8951), and the deruxtecan is conjugated to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-high-expressing tumors via the glycine-glycine-phenylalanine-glycine tetrapeptide-based cleavable linker. In some embodiments of the ADC comprising the ADC drug-linker conjugate deruxtecan, the drug-to-antibody ratio (DAR) is 8. In certain embodiments of the provided treatment methods, the cancer is primary AML. In some embodiments, the cancer is primary MCL.

[0080] The present disclosure provides methods for modulating a CD180-mediated immune response in a subject, comprising administering to the subject any one of the ADCs described and provided herein. In one embodiment, the CD180-mediated immune response is B cell antigen-presenting cell activity, antibody secretion, or proliferation of B lymphocytes, monocytes, or dendritic cells. In some embodiments, modulating a CD180-mediated immune response comprises enhancing the CD180-mediated immune response or inhibiting the CD180-mediated immune response.

[0081] The present disclosure provides methods of treating a disease in a subject, comprising administering to the subject any of the ADCs provided herein. In some embodiments, the disease is a viral infection, a bacterial infection, an autoimmune disease, or an immune disorder.

[0082] The present disclosure provides an isolated anti-CD180 binding molecule comprising a set of three heavy chain complementarity determining regions, HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity determining regions, LCDR1, LCDR2, and LCDR3, wherein the set of HCDR1, HCDR2, and HCDR3 and the corresponding set of LCDR1, LCDR2, and LCDR3 each comprise the following amino acid sequences: HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and SEQ ID NOs: 4, 5 and 15; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and SEQ ID NOs: 4, 5, and 45; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 54 to 56 and SEQ ID NOs: 57 to 59; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 68-70 and SEQ ID NOs: 57, 58, and 71; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and SEQ ID NOs: 80-82; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and SEQ ID NOs: 91-93; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 102, and 25 and SEQ ID NOs: 4, 103, and 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and SEQ ID NOs: 112, 5, and 113; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and SEQ ID NOs: 4, 5, and 130; SEQ ID NOs: 1, 24, and 25 and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 4 to 6.

[0083] In one embodiment of the ADC, the anti-CD180 binding molecule comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and light chain variable region comprise the amino acid sequence of SEQ ID NOs: 7 to 8, 16 to 17, 27 to 28, 36 to 37, 46 to 47, 60 to 61, 72 to 73, 83 to 84, 94 to 95, 104 to 105, 114 to 115, 122 to 123, 131 to 132, or 139 to 140.

[0084] In some ADC embodiments, the anti-CD180 binding molecule comprises a heavy chain and a light chain, and the heavy chain and light chain comprise the amino acid sequence of SEQ ID NOs: 11-12, 20-21, 31-32, 40-41, 50-51, 64-65, 76-77, 87-88, 98-99, 108-109, 118-119, 126-127, 135-136, or 143-144.

[0085] In certain embodiments of the anti-CD180 binding molecule, the binding molecule comprises an IgG, Fv, scFv, Fab, F(ab'), minibody, diabody, triabody, nanobody, bispecific antibody, single domain antibody, or chimeric antigen receptor. In some embodiments, the binding molecule is an IgG, Fv, scFv, Fab, F(ab'), minibody, diabody, triabody, nanobody, bispecific antibody, single domain antibody, or chimeric antigen receptor. In one embodiment of the anti-CD180 binding molecule, the IgG is IgG1, IgG2, IgG3, or IgG4. In one embodiment, the bispecific anti-CD180 antibody is a biparatopic antibody, i.e., a bispecific antibody that targets two non-overlapping epitopes on the same target antigen. In some embodiments, the bispecific antibody binds to CD180 and CD123 (a "CD180xCD123 bispecific antibody"). In various embodiments, the bispecific anti-CD180 antibody comprises the variable heavy chain (VH) region and variable light chain (VL) region and / or CDRs disclosed herein, and a combination of VH, VL, and / or CDRs disclosed herein, i.e., a set of three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, each of which comprises the amino acid sequences disclosed herein, and the corresponding set of LCDR1, LCDR2, and LCDR3.

[0086] Also provided herein are isolated polynucleotide sequences encoding the anti-CD180 binding molecules described herein. In one embodiment, a vector comprises the isolated polynucleotide sequence. In some embodiments, a host cell comprises the described vector.

[0087] Also provided herein are compositions comprising any one of the anti-CD180 binding molecules provided herein and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises an ADC, wherein the ADC comprises a monoclonal antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor, wherein the monoclonal antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor is an anti-CD180 antibody or antigen-binding fragment thereof comprising a set of three heavy chain complementarity-determining regions, HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity-determining regions, LCDR1, LCDR2, and LCDR3, wherein the set of HCDR1, HCDR2, and HCDR3 and the corresponding set of LCDR1, LCDR2, and LCDR3 each comprise the following amino acid sequences: HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5 and 15; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 45; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 54 to 56 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57 to 59; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 68 to 70 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57, 58, and 71; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 80-82; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 91-93; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 102, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 103, and 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 112, 5, and 113; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 130; or HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6.

[0088] Also provided herein is a composition comprising any one of the ADCs provided herein and a pharmaceutically acceptable carrier. The composition comprises an ADC comprising a monoclonal antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor, wherein the monoclonal antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor is an anti-CD180 antibody or antigen-binding fragment thereof comprising a set of three heavy chain complementarity-determining regions, HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity-determining regions, LCDR1, LCDR2, and LCDR3, wherein the set of HCDR1, HCDR2, and HCDR3 and the corresponding set of LCDR1, LCDR2, and LCDR3 each comprise the following amino acid sequences: HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5 and 15; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 45; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 54 to 56 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57 to 59; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 68 to 70 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57, 58, and 71; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 80-82; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 91-93; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 102, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 103, and 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 112, 5, and 113; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 130; or HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6.

[0089] Further provided herein are compositions comprising a polynucleotide sequence encoding the above-described anti-CD180 binding molecules. In one embodiment, the polynucleotide sequence comprises an expression vector for expressing the anti-CD180 binding molecule in a cell.

[0090] The present disclosure provides a method for regulating a CD180-mediated immune response in a subject, comprising administering any of the compositions provided herein to the subject. In one embodiment, the CD180-mediated immune response is B cell antigen-presenting cell activity, antibody secretion, or proliferation of B lymphocytes, monocytes, or dendritic cells. In some embodiments, regulating a CD180-mediated immune response includes enhancing a CD180-mediated immune response or inhibiting a CD180-mediated immune response.

[0091] The present disclosure provides a method for treating a disease in a subject, comprising administering any of the compositions provided herein to the subject.In one embodiment, the disease is a viral infection, a bacterial infection, cancer, an autoimmune disease, or an immune disorder.In a particular embodiment, the disease is a CD180-mediated disease.In one embodiment, the cancer is acute myeloid leukemia (AML), mantle cell lymphoma (MCL), multiple myeloma, follicular lymphoma, acute B-lymphoblastic leukemia, or diffuse large B-cell lymphoma.

[0092] The present disclosure provides a method for treating a cancer that highly expresses CD180 in a subject, comprising administering to the subject any one of the compositions provided herein, comprising one of the described ADCs and a pharmaceutically acceptable carrier. In one embodiment of the provided method, the ADC is an anti-CD180 antibody or antigen-binding fragment thereof that comprises a monoclonal antibody or antigen-binding fragment thereof that targets a tumor that highly expresses CD180, and that comprises a set of three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3; The set of HCDR1, HCDR2 and HCDR3 and the corresponding set of LCDR1, LCDR2 and LCDR3 each comprise the following amino acid sequences: HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5 and 15; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and LCDR1, LCDR2 and LCDR of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 45; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 54 to 56 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57 to 59; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 68 to 70 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57, 58, and 71; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 80-82; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 91-93; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 102, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 103, and 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 112, 5, and 113; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 130; or HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6.

[0093] The present disclosure provides methods of treating a cancer that highly expresses CD180 in a subject, the method comprising administering to the subject any one of the compositions described herein, comprising any one of a monoclonal antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor, a cytotoxic drug payload, and an ADC comprising a linker moiety conjugating the antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor to the cytotoxic drug payload, wherein the cytotoxic drug payload comprises the ADC drug-linker conjugate tesirin (SG3249), wherein tesirin comprises the cytotoxic drug payload, and the cytotoxic drug payload comprises a pyrrolobenzodiazepine dimer cytotoxic The present invention also includes a DNA alkylating agent (SG3199), wherein tesirin is conjugated via a cleavable linker moiety to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors, and the monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors is an anti-CD180 antibody or antigen-binding fragment thereof that comprises a set of three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein the set of HCDR1, HCDR2, and HCDR3 and the corresponding set of LCDR1, LCDR2, and LCDR3 each comprise the following amino acid sequences: HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5 and 15; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 45; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 54 to 56 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57 to 59; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 68 to 70 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57, 58, and 71; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 80-82; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 91-93; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 102, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 103, and 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 112, 5, and 113; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 130; or HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6.

[0094] In one particular embodiment, cancer is acute myeloid leukemia (AML), mantle cell lymphoma (MCL), multiple myeloma, follicular lymphoma, acute B-lymphoblastic leukemia or diffuse large B-cell lymphoma.In one embodiment, cancer is primary AML.In some embodiments, cancer is primary MCL.

[0095] The present disclosure provides a method of treating a cancer that highly expresses CD180 in a subject, comprising administering to the subject a composition ADC comprising the ADC drug-linker conjugate deruxtecan, wherein the deruxtecan comprises a cleavable linker, a self-immolative aminomethylene spacer, and a cytotoxic drug payload, wherein the cytotoxic drug payload comprises a topoisomerase 1 inhibitor payload, wherein the topoisomerase 1 inhibitor payload is a derivative of exatecan (DX-8951), and wherein deruxetan is conjugated to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-high expressing tumors via a cleavable linker, wherein the cleavable linker is a glycine-glycine-phenylalanine-glycine tetrapeptide-based linker. In one embodiment of the provided treatment method, the ADC comprises a monoclonal antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor, and the monoclonal antibody or antigen-binding fragment thereof that targets a CD180-highly expressing tumor is an anti-CD180 antibody or antigen-binding fragment thereof comprising a set of three heavy chain complementarity-determining regions, HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity-determining regions, LCDR1, LCDR2, and LCDR3, and the set of HCDR1, HCDR2, and HCDR3 and the corresponding set of LCDR1, LCDR2, and LCDR3 each comprise the following amino acid sequences: HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1 to 3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5 and 15; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 45; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 54 to 56 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57 to 59; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 68 to 70 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57, 58, and 71; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 80-82; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 91-93; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 102, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 103, and 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 112, 5, and 113; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 130; or HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6.

[0096] In some embodiments of the provided method of treatment, cancer is acute myeloid leukemia (AML), mantle cell lymphoma (MCL), multiple myeloma, follicular lymphoma, acute B-lymphoblastic leukemia or diffuse large B-cell lymphoma.In certain embodiments, cancer is primary AML.In certain embodiments of the described method, cancer is primary MCL.

[0097] Method used As used herein, the term "method" refers to any manner, means, technique, or procedure for accomplishing a given task, including, but not limited to, any manner, means, technique, or procedure that is known in the chemical, pharmacological, biological, biochemical, or medical arts or that can be readily developed from known manners, means, techniques, and procedures by a practitioner in the chemical, pharmacological, biological, biochemical, or medical arts.

[0098] As used herein, "modulating" refers to "stimulating" or "inhibiting" the activity or pathway of a molecular target. For example, a composition modulates the activity or pathway of a molecular target if it stimulates or inhibits the activity or pathway of the molecular target by at least 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or about 99% or more, compared to the activity or pathway of the molecular target under the same conditions but lacking the presence of the composition. In another example, a composition modulates the activity or pathway of a molecular target if it stimulates or inhibits the activity or pathway of the molecular target by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold, compared to the activity or pathway of the molecular target under the same conditions but lacking the presence of the composition. The activity or pathway of a molecular target may be measured by any reproducible means. The activity or pathway of a molecular target may be measured in vitro or in vivo. For example, the activity or pathway of a molecular target may be measured in vitro or in vivo by a suitable assay known in the art for measuring activity. A control sample (not treated with the composition) may be assigned a relative activity value of 100%.

[0099] In one embodiment, the present disclosure provides a method of modulating a CD180-mediated immune response in a subject, comprising administering to the subject a composition comprising any one of the anti-CD180 binding molecules disclosed herein. In one embodiment, the composition comprises an antibody-drug conjugate disclosed herein. In one embodiment, the antibody-drug conjugate comprises tesirin. In another embodiment, the present disclosure provides a method of modulating a CD180-mediated immune response in a subject, comprising administering to the subject a composition comprising any one of the polynucleotide sequences disclosed herein.

[0100] In one embodiment, the CD180-mediated immune response comprises antigen-presenting cell activity of cells of the B cell lineage. In another embodiment, the CD180-mediated immune response comprises antibody secretion. In another embodiment, the CD180-mediated immune response comprises proliferation of B lymphocytes, monocytes, and / or dendritic cells.

[0101] In one embodiment, the modulating method comprises stimulating antigen-presenting cell activity of cells of the B cell lineage. In another embodiment, the modulating method comprises inhibiting antigen-presenting cell activity of cells of the B cell lineage. In one embodiment, the modulating method comprises stimulating antibody secretion (e.g., by B cells). In another embodiment, the modulating method comprises inhibiting antibody secretion (e.g., by B cells). In one embodiment, the modulating method comprises stimulating proliferation of B lymphocytes, monocytes and / or dendritic cells. In another embodiment, the modulating method comprises inhibiting proliferation of B lymphocytes, monocytes and / or dendritic cells.

[0102] In another embodiment, the present disclosure provides a method of treating a disease in a subject, comprising administering to the subject a composition comprising any one of the anti-CD180 binding molecules disclosed herein. In another embodiment, the present disclosure provides a method of treating a disease in a subject, comprising administering to the subject a composition comprising any one of the polynucleotide sequences disclosed herein. In one embodiment, the disease comprises any cancer or tumor cell that expresses CD180. In another embodiment, the disease comprises any disease the etiology or treatment of which involves the function or activity of antigen-presenting cells. In another embodiment, the disease comprises any disease the etiology or treatment of which involves antibody secretion by B cells. In one embodiment, examples of cancers or diseases include, but are not limited to, acute myeloid leukemia, mantle cell lymphoma, multiple myeloma, follicular lymphoma, acute B-lymphoblastic leukemia, and diffuse large B-cell lymphoma.

[0103] In another embodiment, the anti-CD180 binding molecules or anti-CD180 antibodies disclosed herein can be used as immunotherapeutics to enhance or inhibit the activity of B cells as antigen-presenting cells. In another embodiment, the anti-CD180 binding molecules or anti-CD180 antibodies disclosed herein can be used as immunotherapeutics to enhance or inhibit antibody secretion by B cells. In one embodiment, the immunotherapeutics are useful in relation to cancers or diseases including, but not limited to, acute myeloid leukemia, mantle cell lymphoma, multiple myeloma, follicular lymphoma, acute B-lymphoblastic leukemia, and diffuse large B-cell lymphoma.

[0104] As used herein, the terms "treat," "treatment," or "therapy" (and variations thereof) refer to therapeutic treatment and include prophylactic and preventative measures, the purpose of which is to prevent or slow (alleviate) undesirable physiological changes associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of the disease or condition, stabilization of the disease or condition (i.e., where the disease or condition does not worsen), delay or slowing of the progression of the disease or condition, improvement or palliation of the disease or condition, and remission (whether partial or complete) of the disease or condition. Subjects in need of treatment include those already suffering from a disease or condition as well as those susceptible to a disease or condition in need of prevention.

[0105] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a human or non-human animal to which treatment with the compositions or formulations of the present disclosure is provided. The terms "non-human animal" and "non-human mammal" are used interchangeably herein and include all vertebrates, including mammals such as non-human primates (e.g., higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, horses, and non-mammals such as reptiles, amphibians, chickens, and turkeys. The compositions described herein can be used to treat any suitable mammal, including primates (such as monkeys and humans), horses, cows, cats, dogs, rabbits, and rodents (such as rats and mice). In one embodiment, the mammal to be treated is a human. The human can be any human of any age. In one embodiment, the human is an adult. In another embodiment, the human is a child. The human may be male, female, pregnant, middle-aged, adolescent, or elderly.

[0106] Suitable pharmaceutical compositions for use in the methods disclosed herein include compositions in which the active ingredients are contained in an effective amount to achieve the intended purpose. In one embodiment, a therapeutically effective amount refers to an amount of one or more active ingredients (e.g., an anti-CD180 binding molecule or an anti-CD180 antibody) that is effective to prevent, alleviate, or ameliorate symptoms of disease, or to prolong the survival of a subject being treated. Determining a therapeutically effective amount is well within the capabilities of one of ordinary skill in the art.

[0107] In one embodiment, the exact amount of the polypeptide or composition thereof herein required to achieve the desired effect will vary from subject to subject, depending on the species, age, sex, weight, and general condition of the subject, the specific polypeptide, the route of administration, and whether other drugs are included in the regimen. Therefore, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using routine testing. Dosages can vary, and the polypeptide can be administered in one or more daily doses (e.g., two or more, three or more, four or more, or five or more) per day for one or more days. Guidance for selecting the appropriate dose for an antibody can be easily found in the literature.

[0108] In one embodiment, the disease can be a viral infection, a bacterial infection, cancer, an autoimmune disease, or an immune disorder. In one embodiment, the disease can be a viral infection of the upper respiratory tract, an early pulmonary infection, or a late pulmonary infection. Many diseases and cancers are known to be caused by viruses. Examples of disease-causing viruses include, but are not limited to, norovirus; rotavirus; hepatitis A, B, C, D, or E virus; rabies virus, West Nile virus, enterovirus, echovirus, coxsackievirus, herpes simplex virus (HSV), HSV-2, varicella-zoster virus, mosquito-borne virus, arbovirus, St. Louis encephalitis virus, California encephalitis virus, lymphocytic choriomeningitis virus, human immunodeficiency virus (HIV), poliovirus, Zika virus, rubella virus, cytomegalovirus, human papillomavirus (HPV), enterovirus D68, severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome coronavirus, SARS coronavirus 2, Epstein-Barr virus, influenza virus, respiratory syncytial virus, polyomavirus (such as JC virus, BK virus), Ebola virus, dengue virus, or any combination thereof.

[0109] In another embodiment, the disease is cancer, including carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, blastoma, chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone, osteosarcoma, rhabdomyosarcoma, heart cancer, brain tumor, astrocytoma, glioma, medulloblastoma, neuroblastoma, breast cancer, medullary carcinoma, adrenocortical cell carcinoma, thyroid cancer, Merkel cell carcinoma, eye cancer, cancer of the gastrointestinal tract, colon Cancer can include, but is not limited to, gallbladder cancer, gastric (or stomach) cancer, gastrointestinal carcinoid tumor, hepatocellular carcinoma, pancreatic cancer, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, renal cell carcinoma, prostate cancer, testicular cancer, urinary tract cancer, uterine sarcoma, vaginal cancer, head cancer, neck cancer, nasopharyngeal cancer, hematopoietic cancer, non-Hodgkin's lymphoma, skin cancer, basal cell carcinoma, melanoma, small cell lung cancer, non-small cell lung cancer, or any combination thereof.

[0110] In another embodiment, the disease is an autoimmune disease, such as achalasia, amyloidosis, ankylosing spondylitis, anti-glomerular basement membrane / anti-tubular basement membrane (anti-GBM / anti-TBM) nephritis, antiphospholipid syndrome, arthritis, autoimmune angioedema, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, Behcet's disease, celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy, Cogan's syndrome, congenital heart block, Crohn's disease, dermatitis, dermatomyositis, discoid lupus erythematosus, Dressler's syndrome, endometriosis, fibromyalgia, fibrosing alveolitis, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, herpes gestationis, immune thrombocytopenia Hypoplastic purpura, interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, lichen planus, lupus erythematosus, Lyme disease, multiple sclerosis, myasthenia gravis, myositis, neonatal lupus, neutropenia, relapsing rheumatoid arthritis, peripheral neuropathy, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome The disease may be, but is not limited to, primary biliary cirrhosis, primary sclerosing cholangitis, progestational dermatitis, psoriasis, psoriatic arthritis, reactive arthritis, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, thrombocytopenic purpura, type 1 diabetes, ulcerative colitis, uveitis, vasculitis, and vitiligo.

[0111] In some embodiments, the disease is a transplant-related disease, such as graft-versus-host disease (GvHD). In one embodiment, the GVHD is acute GVHD. In another embodiment, the GVHD is chronic GVHD.

[0112] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims below find experimental support in the following examples.

[0113] Example Example 1: Immune Fab-phage library construction and identification of lead anti-CD180 antibodies Using our proprietary Alloy Fab-phage display vector, an immune Fab-phage display library was generated from B cell RNA from Alloy ATX-Gk® mice immunized with human CD180 extracellular domain (ECD) HIS-tagged protein (Sino Biological, 11370-H08H; Creative Biomart, CD180-3900H). A second immune Fab-phage display library was generated from B cell RNA from Alloy ATX-Gk® mice immunized with human CD180 / human MD-1 DNA (pDUO-hMD1 / RP105, InvivoGen, pduo-hmd1rp105). Both Fab-phage libraries were confirmed to consist of Fabs containing full-length VH / VK pairs by Sanger sequencing (Sanger et al., Proc. Natl. Acad. Sci. 1977;74:5463).

[0114] Fab phages from each library were enriched twice for Fab-specific CD180 (ECD) binders by panning against recombinant CD180 (ECD)-HIS protein. For protein panning, CD180 (ECD)-binding Fab phages were enriched using biotinylated human or rhesus monkey CD180 (ECD)-HIS protein (Sino Biological, 11370-H08H; Creative Biomart, CD180-1598R) and streptavidin magnetic beads (Invitrogen, 60210). To ensure that the captured Fab-phages were specific for CD180, protein panning with biotinylated CD180 (ECD) protein was performed in the presence of at least a 5-fold molar excess of a soluble CD180-like protein competitor complex consisting of human TLR4 (ECD)-HIS / MD-2_HIS (R&D Systems, 3146-™). The ECD of TLR4 shares 28.2% sequence identity with human CD180 (ECD). In each round of panning, Fab phages bound to CD180 (ECD)-biotin-streptavidin-beads were eluted with 100 mM triethylamine (TEA; Sigma-Aldrich, T0886) and then neutralized with 1 M Tris, pH 8.0 (Invitrogen, AM9855G).

[0115] Cell panning was also performed two to three times with Fab phages from each library using HEK293 cells overexpressing human CD180 / human MD-1 (pDUO-hMD1 / RP105, InvivoGen, pduo-hmd1rp105) (Nagai Y. et al., 2002, Blood, 99(5):1699). Prior to panning, the Fab phage library was depleted with HEK293 cells overexpressing human MD-1 (pUN01-hMD1, InvivoGen, punol-hmd1) to remove human MD-1 cross-reactive Fabs. For each round of panning, Fab phages that did not bind to MD-1 cells were panned against HEK293 cells overexpressing human CD180 / human MD-1, and cell-bound Fab phages were eluted with 100 mM TEA and neutralized with 1 M Tris, pH 8.0.

[0116] Protein and cell ELISAs of polyclonal Fab-phages isolated by protein and cell panning confirmed that Fab-phages were preferentially enriched for the panning reagents; i.e., Fab-phages isolated by human CD180(ECD)-HIS protein panning bound more strongly to recombinant proteins than to human CD180 / MD-1-overexpressing HEK293 cells. Furthermore, ELISAs using plate-immobilized recombinant proteins confirmed that panned Fab-phages were enriched up to 40-fold for human CD180(ECD)-HIS and lacked reactivity with the human TLR4 / MD-2 protein complex or human MD-1-Fc (BON-OPUS, CJ56).

[0117] To confirm the specific enrichment of polyclonal Fab-phages against the target CD180, 1,920 monoclonal Fabs were expressed by infecting and propagating panned phages from the two libraries in Escherichia coli SS320, a bacterial strain that recognizes the amber stop codon located between the Fab and the phage coat protein g3p in the phage vector (Sidhu et al., Methods in Enzymology, 2000;328:333). Protein ELISA and cell ELISA results on the monoclonal Fab supernatants, generated using Triton® X-100 (Sigma-Aldrich, T9284-1L) in induction medium, confirmed that 75% (1,438) of the Fabs were positive for the target CD180. Sanger sequencing of CD180-binding monoclonal Fabs confirmed a high degree of antibody diversity, consisting of 134 HCDR3 clonotypes (each consisting of Fabs with at least 80% HCDR3 sequence identity), 24 VH genes, and 16 VK genes. 219 Fabs representing representative HCDR3 clonotype and VH / VK gene diversity were purified using HisPur® Ni-NTA magnetic beads (Thermo Scientific, 88832) and tested for specificity against the target CD180 using ELISA and flow cytometry. Based on the latter results, 48 ​​lead Fabs were selected for reformulation as IgG1s for further characterization of their anti-CD180 activity.

[0118] Generation of an antibody panel against CD180 To generate antibodies against CD180, two cohorts, each containing five ATX-GK mice, were immunized. ATX-GK mice are capable of producing human antibodies. Cohort 1 was immunized subcutaneously weekly with His-tagged CD180 protein (Sino Biological, 11370-H08H; Creative Biomart, CD180-3900H), while cohort 2 was immunized intradermally weekly with plasmid DNA containing human CD180 / human MD-1 (pDUO-hMD1 / RP105, InvivoGen, pduo-hmd1rp105). After a total of five injections for cohort 1 and eight injections for cohort 2, the mice were sacrificed, and their splenocytes were used for hybridoma generation and immune phage library construction.

[0119] For titer measurements, mouse serum was prepared and used in an ELISA for CD180 protein (Titer Assay #1) or a flow cytometric cell-based binding assay (Titer Assay #2). Briefly, for Titer Assay #1, ELISA plates were coated with CD180 protein. After blocking the plates with 3% BSA buffer for 1 hour and washing with PBS, serum dilutions were transferred to the ELISA plates and incubated for 1 hour. After washing with PBS, a secondary antibody specific for mouse IgG was added to the ELISA plates for 1 hour. After washing with PBS, TMB solution was added, and the reaction was then stopped with sulfuric acid.

[0120] For titer assay #2, serum dilutions were incubated with cell lines expressing the CD180 / MD1 complex for 1 hour. After washing with PBS, a secondary antibody specific for mouse IgG was added to the plate, and mean fluorescence intensity was measured using flow cytometry.

[0121] To generate hybridomas, splenocytes were fused with myeloma cell lines using standard protocols to generate hybridomas (Kohler and Milstein, Nature, 1975, 256:495). Individual hybridoma supernatants were screened by ELISA for binding to human CD180. Positive clones were then tested by flow cytometry for binding to cells expressing CD180 to select hybridomas reactive with native CD180.

[0122] Table 1 shows the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) for the various antibodies provided herein. Table 2 shows the light chain CDR sequences (LCDR1, LCDR2, LCDR3) for the various antibodies provided herein.

[0123] Table 3 shows the amino acid sequences of the heavy chain variable region (VH), light chain variable region (VL), heavy chain (HC), and light chain (LC) for the various antibodies described herein. Table 4 shows the nucleotide sequences encoding the VH, VL, HC, and LC for the various antibodies described herein.

[0124] [Table 1]

[0125] [Table 2]

[0126] [Table 3] TIFF2026505787000005.tif233159TIFF2026505787000006.tif237159TIFF2026505787000007.tif241159TIFF2026505787 000008.tif234159TIFF2026505787000009.tif234159TIFF2026505787000010.tif233159TIFF2026505787000011.tif36159

[0127] [Table 4] TIFF2026505787000013.tif241159TIFF2026505787000014.tif239159TIFF2026505 787000015.tif236159TIFF2026505787000016.tif238159TIFF2026505787000017.t if238159TIFF2026505787000018.tif240159TIFF2026505787000019.tif237159TIF F2026505787000020.tif236159TIFF2026505787000021.tif238159TIFF2026505787 000022.tif237159TIFF2026505787000023.tif241159TIFF2026505787000024.tif2 40159TIFF2026505787000025.tif240159TIFF2026505787000026.tif239159TIFF20 26505787000027.tif241159TIFF2026505787000028.tif241159TIFF2026505787000 029.tif241159TIFF2026505787000030.tif241159TIFF2026505787000031.tif44159

[0128] Example 2: Characterization of anti-CD180 antibodies Cell ELISA assay All mAbs were subjected to cell-binding screening using a flow cytometer. Engineered cells expressing CD180 / MD-1, MD-1, or parental HEK cells were used to test antibody binding. Antibodies were incubated with cells at 100 nM or 30 nM concentrations for 45 minutes at 4°C. R-phycoerythrin AffiniPure goat anti-human IgG (Jackson Immunoresearch 109-115-098) was used as the secondary antibody. Antibody binding on cells was detected using an Intellicyt iQue3 VBR. Figure 1A shows that all CHAMP-mAbs had significant binding in the ELISA. These antibodies did not bind to cells expressing MD-1 alone (Figure 1B).

[0129] EC 50 evaluation For EC50, anti-CD180 antibodies were tested at concentrations ranging from 100 nM to 0.6 pM (serial 3-fold dilutions) for binding on CD180 / MD-1 cells. Cells were then incubated with the secondary antibody R-Phycoerythrin AffiniPure Goat Anti-Human IgG (Jackson Immunoresearch 109-115-098). Data were acquired on an Intellicyt iQue3 VBR. Median fluorescence intensity (MFI) was plotted against the concentration of anti-CD180 antibody. EC50 values ​​were obtained by fitting a four-parameter dose-response curve. The results in Figure 2 show the quantitative binding affinity of the antibodies to CD180, demonstrating specific and potent binding.

[0130] Surface plasmon resonance (SPR) for binding affinity Surface plasmon resonance (SPR) was used to identify intrinsic binding affinities. Kinetic experiments were performed on a Carterra LSA using running buffer (PBS (pH 7.40), 1% BSA, 0.05% Tween 20). Antibodies were captured on an anti-human Fc capture chip prepared with an HC30M chip. For kinetic analysis, purified recombinant His-tagged human CD180 (Sino Biologics or Creative Biomart) or His-tagged rhesus monkey CD180 (Creative Biomart) was injected sequentially at concentrations ranging from 0.076 nM to 1500 nM (3-fold serial dilutions). For each concentration, 5 min of binding was followed by 15 min of dissociation. Results were processed and analyzed using Carterra LSA Kinetics software. The kinetic data were referenced to an interstitial reference spot and double-referenced to a buffer cycle, then globally fit to a 1:1 binding model to determine their apparent association and dissociation rate constants (k on and k off The value of k was determined. off / k on The ratio of these two values ​​was used to calculate the KD value of each antigen / mAb interaction, i.e., KD = k off / k on was derived.

[0131] Figures 3A-3C show that only Champ mAb-006 and Champ mAb-007 exhibited binding to soluble CD180 protein, suggesting that the epitopes for all other Champ mAbs are masked when CD180 is expressed as a soluble protein.

[0132] DSF and turbidity assays DSF and turbidity assays were used to measure the thermal aggregation rate of the antibodies. Thermal stability was assessed by nano-differential scanning fluorimetry (nanoDSF) on a Prometheus Panta. Each sample was measured in duplicate. The antibody melting temperature was detected during heating with a linear thermal ramp (0.5°C / min, 25-95°C). Data were analyzed using Panta Analysis software. The unfolding transition points were determined from the change in the emission wavelength of tryptophan fluorescence at 350 nm and 330 nm. Figure 4 shows that all antibodies exhibited acceptable melting temperatures (Tm) above 65°C. Thermal stability is a general biophysical property of antibodies that should be accounted for in lead selection. All lead antibodies should have a thermal stability above 65°C. The first peak (TM1) is the unfolding of the VL, and the second peak (TM2) is the unfolding of the VH / CH.

[0133] Affinity Capture Self-Interacting Nanoparticle Spectroscopy (AC-SINS) Assay The possibility of antibody self-interaction was tested using AC-SINS. Gold nanoparticles (Ted Pella, 15705-20) were washed with water. An 80 / 20 (v / v) mixture of capture / non-capture antibodies (Jackson Immuno Research Labs) was buffer-exchanged into 20 mM sodium acetate (pH 4.5) to a concentration of 500 μg / ml. To prepare 1 ml of coated particles, 900 μL of gold nanoparticles were incubated overnight with 100 μL of the antibody mixture for 90 minutes at room temperature. After antibody coating, the beads were quenched with thiolated PEG (MW: 2000 Da). The beads were then concentrated 10x in PBS. 10 μL of the 10x concentrated particle solution was incubated with 100 μL of a 40 μg / mL antibody sample in a 384-well polypropylene plate for 2 hours at room temperature. Plates were then immediately spun down at 3000 rpm and scanned from 510 to 580 nm in 2 nm increments on a Synergy Neo2 Multi-Mode Plate Reader (BioTek). Reported values ​​are the average of duplicate wells and are the red-shifted wavelength of the sample at maximum absorbance minus the blank reference value (PBS only). A larger red-shift indicates enhanced self-interaction.

[0134] FIG. 5 shows that all CHAMP antibodies exhibited a favorable profile of shifts less than 11 nM, suggesting a low propensity for self-interaction.

[0135] Size exclusion chromatography Size exclusion chromatography, also known as molecular sieve chromatography, is a chromatographic method in which molecules in a solution are separated by their size, and in some cases, molecular weight. Size exclusion chromatography (SEC) was performed on an Agilent 1200 series HPLC instrument using a YMC Diol-200 8 x 300 mm column (catalog number: DL20S05-3008WT). The running buffer was 20 mM sodium phosphate, 400 mM NaCl (pH 7.0), and the flow rate was 0.3 mL / min. For freeze / thaw stability, samples were frozen at -80°C for 20 minutes and then thawed at room temperature for approximately 20 minutes.

[0136] FIG. 6 shows that all CHAMP antibodies exhibit high levels of purity (>95%) after production both before and after freeze-thaw cycles (1× F / T).

[0137] Capillary electrophoresis sodium dodecyl sulfate (CE-SDS). CE-SDS is an analytical method used to assess protein purity. CE-SDS using the Protein Express Assay Reagent Kit (Perkin Elmer, #CLS960008) was performed on a LapChip GX II device using the Protein Express 200 (Perkin Elmer, #760499). The reagents and chips were prepared according to the manufacturer's instructions. Briefly, reducing buffer was prepared by mixing 1 M dichlorodiphenyltrichloroethane with Protein Express Sample Buffer, while non-reducing buffer consisted of Protein Express Sample Buffer alone. Samples were mixed with reducing or non-reducing buffer and denatured at 80°C for 10 minutes. Samples were centrifuged at 2,000 g for 1 minute to remove air bubbles and then placed in the LapChip GX II device for analysis.

[0138] FIG. 7 shows that all CHAMP mABs tested by this method exhibit high levels of purity (>95%) after production.

[0139] Baculovirus particle ELISA Baculovirus particle ELISA was used to test the tendency of antibodies to interact with proteins in a nonspecific manner. The method was similar to that reported by Hotzel et al. (2012). Briefly, baculovirus particles (BVP, Lake Pharma) were diluted 1:100 in 50 mM sodium bicarbonate (pH 9.3). After overnight incubation of 50 μL of BVP on an ELISA plate (3369; Corning) at 4°C, unbound BVP was aspirated from the wells. All remaining steps were performed at room temperature. The plate was blocked with 100 μL of blocking buffer (PBS containing 1% BSA) for 1 hour and then washed three times with 100 μL of PBS. 50 μL of 16 nM test antibody was then added to the wells, incubated for 1 hour, and then washed with 100 μL of PBS. HRP-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch) was used as the secondary antibody and incubated for 1 hour, followed by washing as before. Finally, 50 μL of TMB substrate (34021; Fisher Scientific) was added to each well and incubated for 10–15 minutes. The reaction was stopped by adding 50 μL of 2 M sulfuric acid to each well. Absorbance was read at 450 nm, and BVP scores were determined by normalizing absorbance to control wells without the test antibody.

[0140] FIG. 8 shows that all CHAMP mAbs exhibit a low tendency for nonspecific and polyreactive binding.

[0141] Example 3: Antibody Drug Conjugates (ADCs) Antibody / drug conjugates (ADCs) are a newly emerging class of highly potent pharmaceuticals that represent an exceptional combination of chemotherapy and immunotherapy. The concept of ADCs was first proposed nearly 100 years ago by German physician and scientist Paul Ehrlich. Ehrlich described antibodies as "magic bullets" that find their targets on their own without causing any harmful effects to the organism. Ehrlich also predicted the attachment of toxins to antibodies to improve their therapeutic specificity.

[0142] Currently, antibody-drug conjugates (ADCs) are one of the most rapidly growing anticancer drugs. In one embodiment, this approach involves a monoclonal antibody (mAb) conjugated to a cytotoxic payload via a chemical linker. ADCs selectively target specific antigens on cancer cells with highly potent cytotoxic agents, providing maximum efficacy while minimizing systemic toxicity. The selection of the appropriate target, mAb, cytotoxic payload, and how the antibody is linked to the payload are key determinants of the safety and efficacy of ADCs.

[0143] Tesirin (SG3249) is an antibody-drug conjugate pyrrolobenzodiazepine (PBD) dimer payload. Tesirin combines potent antitumor activity with desirable physicochemical properties, such as favorable hydrophobicity and improved conjugation reactivity. SG3199 is the released warhead component of the ADC payload Tesirin. SG3199 retains picomolar-level activity in a panel of cancer cell lines. The PBD dimer is a highly efficient DNA minor groove crosslinker with potent cytotoxicity.

[0144] In one embodiment, Ab-001 and monoclonal antibody Ab-014 hIgG1, as well as a negative control IgG1 mAb, were conjugated to the linker-payload tesirine via stochastic maleimide conjugation to the interchain cysteines to generate CO-ADC-001 and CO-ADC-002 (Figure 9). Nonspecific drug conjugation on naturally available reduced interchain cysteine ​​residues is used to establish a chemical link between the antibody and the payload. The interchain cysteine ​​residues are reduced, followed by the addition of tesirine and re-crosslinking the cysteines. Size exclusion chromatography was performed to confirm the purity of the monomeric ADCs (data not shown).

[0145] Those skilled in the art will be able to easily incorporate the anti-CD180 binding molecules disclosed herein into ADCs containing different linkers and / or payloads. An anti-CD180 ADC containing tesirin is shown herein as an example. Other linkers for ADCs include, but are not limited to, valine-citrulline-PAB or valine-alanine-PAB. Other payloads for ADCs include, but are not limited to, monomethyl auristatin E (MMAE), maytansine, or calicheamicin. Examples of linker-payload combinations include, but are not limited to, tesirin, deruxtecan, ozogamicin, or emtansine.

[0146] The ADCs disclosed herein (e.g., tesirin-conjugated ADCs and deruxtecan-conjugated ADCs) can be used to treat diseases such as, but not limited to, acute myeloid leukemia, mantle cell lymphoma, multiple myeloma, follicular lymphoma, acute B-lymphoblastic leukemia, or diffuse large B-cell lymphoma.

[0147] Figures 10A-10B show the ex vivo cytotoxicity of ADCs in primary acute myeloid leukemia (AML). These results demonstrate effective cell killing in a primary AML model. Cytotoxicity correlates with CD180 expression; CD180 is the target of the antibody in the ADC. The higher the level of CD180 expression, the more potent the ADC.

[0148] Figure 11 shows the inhibition of ex vivo cytotoxicity of ADC in primary acute myeloid leukemia. A naked antibody targeting CD180 was first added to inhibit the binding of the CD180-targeted ADC. As a result, the ADC was unable to bind, thereby eliminating all ADC-mediated cell killing. These results indicate that the cell killing is dependent on ADC binding to CD180.

[0149] Figure 12 shows the ex vivo cytotoxicity of the ADC in primary mantle cell lymphoma (MCL). These results demonstrate effective cell killing in a primary MCL model.

[0150] Example 4: Targeting CD180 in primary AML with anti-CD180 antibody drug conjugates Surface expression of CD180 is enhanced in primary AML cells compared to normal immune cells. Therefore, CD180-highly expressing tumors are targets for treatment with the anti-CD180 antibody-drug conjugates of the present invention.

[0151] Figures 13A-13D show an overview of CD180 targets. Figure 13A shows that CD180 surface expression is enhanced in primary AML. The distribution of protein expression in the CD180 proteomics of primary AML correlates with cell surface expression in primary AML, and CD180 is elevated in primary AML compared to normal immune cells; therefore, cytogenetic abnormalities can be used as a biomarker for first-in-human (FIH) trials. Figure 13B shows CD180 expression in primary cultures of normal bone marrow (BM), suggesting an improved therapeutic index (TI) for CD133 ADCs. CD180 expression is lower than CD133 on normal hematopoietic stem cells (HSCs) and progenitor cells, with expression highest on mature B cells and plasmacytoid dendritic (pDC) cells. Figure 13C shows that CD180 is expressed on leukemic stem cells (LSCs) and progenitor cells in primary AML, which would eliminate measurable residual disease (MRD). Figure 13D shows that enhanced inflammatory signaling is observed in CD180-high tumors. Inflammatory AML provides a survival niche for LSCs. Primary AML with enhanced CD180 expression also shows elevated genes involved in the inflammatory process. Pathways involved in the innate immune response and response to oxidative stress are enhanced in CD180-high primary AML.

[0152] The lead anti-CD180 antibodies, CHAMPmAB-001 and CHAMPmAB-014, were characterized. Figures 14A-14F show the melting temperature, self-interaction, freeze / thaw stability, and purity of the anti-CD180 antibody leads, CHAMPmAB-001 and CHAMPmAB-014: Comparison of CHAMPmAB-001 and CHAMPmAB-014 with isotype controls (ATX-P-84 and mIgG1), with secondary controls (hIgG1, hu2'Ab only, and mu2'Ab only), and with no staining (control sample) for binding to human CD180 / MD1 (positive) cells, MD1 (negative) cells, and parental HEK cells (negative) (Figure 14F).

[0153] The half-maximal effective concentrations (EC) of CHAMPmAB-001 (6 nM) and CHAMPmAB-014 (4 nM) on HEK293 CD180 / MD-1 cells 50 ) is shown in Figure 14G.

[0154] ADCs prepared as described in Example 3 were characterized and conjugated to tesirin or deruxtecan.

[0155] Figures 15A-15C show conjugation of the lead anti-CD180 antibody CHAMPmAB-001 to tesirine compared to conjugation of isotype IgG1 to tesirine. Figure 15A shows a schematic diagram of one embodiment of an ADC of an anti-CD180 antibody conjugated to the linker payload tesirine (x2). Figure 15B shows a drug-to-antibody ratio (DAR) of 2.1; i.e., the average number of drugs conjugated to CHAMPmAB-001, an anti-CD180 antibody of the present disclosure conjugated to the linker payload tesirine (x2) (CO-ADC-001). Figure 15C shows a DAR of 2.2 for isotype IgG1 conjugated to the linker payload tesirine (x2) (CO-ADC-003).

[0156] Figures 16A-16C show the conjugation of the lead anti-CD180 antibody CHAMPmAB-004 to deruxtecan compared to the conjugation of isotype IgG1 to deruxtecan. Figure 16A shows a schematic diagram of one embodiment of an anti-CD180 antibody conjugated to the linker payload deruxtecan (x8). Figure 16B shows the DAR of 8 for the anti-CD180 antibody CHAMPmAB-001 of the present disclosure conjugated to the linker payload deruxtecan (x8). Figure 16C shows the DAR of 8.97 for isotype IgG1 conjugated to the linker payload deruxtecan (x8).

[0157] Example 5: Potent CD180-dependent cytotoxicity of anti-CD180 ADCs was observed in primary AML tumors The cytotoxicity of the anti-CD180 ADC CO-ADC-001, a therapeutic mAb conjugated to tesirin (DAR2), and an isotype control ADC was compared in tumors derived from two primary AMLs (CTG-4044 and CTG-2240) and measured as percent cell viability. ADC CO-ADC-001 was found to have potent CD180-dependent cytotoxicity compared to the isotype control ADC.

[0158] Figure 17A shows that tesirin conjugates are potent ADCs in CTG-2240 primary AML. CO-ADC-001 is a therapeutic anti-CD180 mAb conjugated to tesirin (DAR2). Figure 17B shows that deruxtecan conjugates are also potent ADCs in CTG-2240 primary AML, comparable to MYLOTARG® (gemtuzumab ozogamicin, which is indicated for the treatment of newly diagnosed CD33-positive AML in adults and pediatric patients 1 month of age and older, and for relapsed or refractory CD33-positive AML in adults and pediatric patients 2 years of age and older).

[0159] CO-ADC-004 is a therapeutic anti-CD180 mAb conjugated to deruxtecan (DAR8). Figure 17C shows that the cytotoxicity of CO-ADC-001 correlates with CD180 expression in primary AML. Figure 17D shows that the cytotoxicity results of primary AML suggest a high frequency of response.

[0160] Figures 18A-18C show that blocking CD180 with naked CD180 mAb reduces ADC activity. Figure 18A shows the percent viability of primary AML cells after addition of CO-ADC-001 (300 ng / ml) without first adding a CD180 blocking Ab, compared to the percent viability of primary AML cells after addition of CD180 ADC when 500 ng / ml of naked CD180 antibody was added before addition of the CD180 ADC; the naked antibody inhibited binding of the ADC to CD180. Figure 18B shows the percent viability of primary AML cells after the addition of CO-ADC-002 (300 ng / ml) without an initial CD180 blocking Ab, compared to the percent viability of primary AML cells after the addition of CD180 ADC when naked CD180 antibody (500 ng / ml) was added before the addition of CD180 ADC; the naked antibody inhibited binding of the ADC to CD180. Figure 18C shows the percent viability of primary AML cells after the addition of isotype ADC (300 ng / ml) without an initial CD180 blocking Ab, compared to the percent viability of primary AML cells after the addition of isotype ADC when naked CD180 antibody (500 ng / ml) was added before the addition of CD180 ADC; the naked antibody inhibited binding of the isotype ADC to CD180.

[0161] Example 6: Potent CD180-dependent cytotoxicity of anti-CD180 ADCs observed in primary MCL tumor samples The cytotoxicity of the anti-CD180 ADC CO-ADC-001, a therapeutic mAb conjugated to tesirin (DAR2), and the negative control mAb CO-ADC-003 conjugated to tesirin was compared in tumors derived from three primary MCLs (CTG-3446, CTG-3785, and CTG-3448), measured as percent cell viability. ADC CO-ADC-001 was found to have potent CD180-dependent cytotoxicity compared to the negative control mAb conjugated to tesirin.

[0162] Figures 19A-19C show that CD180 ADCs are potent against primary MCL cells. Figure 19A shows the percent viability of primary MCL cells treated with CO-ADC-001, a therapeutic anti-CD180 mAb conjugated to tesirin, compared with the percent viability of primary MCL cells treated with CO-ADC-003, a negative control mAb conjugated to tesirin. Figure 19A shows the percent viability of primary MCL CTG-3446 cells treated with CO-ADC-001 and CO-ADC-003, respectively. Figure 19B shows the percent viability of primary MCL CTG-3785 cells treated with CO-ADC-001 and CO-ADC-003, respectively. Figure 19C shows the percent viability of primary MCL CTG-3448 cells treated with CO-ADC-001 and CO-ADC-003, respectively.

[0163] Example 7: CD180 ADCs are highly potent against disseminated primary AML in vivo CTG-2240 primary AML tumor cells were injected into mice via TVI (tail vein injection) for engraftment of bone marrow, spleen, and peripheral blood to produce disseminated primary AML cells. Animals were randomized when the bone marrow reached a concentration of 20% AML.

[0164] CO-ADC-004 (anti-CD180 antibody CO-mAb-020 conjugated to deruxtecan) was administered intravenously as a single dose of 5 MKP (mg / kg). Endpoints were assessed by flow cytometry in whole blood and bone marrow.

[0165] The ADC CO-ADC-004 was administered as a single dose of 5 MKP (mg / kg) via intravenous injection. Endpoints were assessed by flow cytometry in bone marrow.

[0166] Figures 20A-20B show that CO-ADC-001, a DAR2 tesirin conjugate targeting CD180 (anti-CD180 antibody CO-mAb-020 conjugated to tesirin), is highly active against disseminated primary AML cells in vivo. Figure 20A shows the characteristics of CTG-2240 primary AML. Figure 20B shows the results of in vivo analysis of tumor (%) in bone marrow, LSC in bone marrow, and CD123 cells in bone marrow after administration of CO-ADC-001 as a single dose of 0.3 MPK (mg / kg) by intravenous injection compared to the 0.3 MPK control and IgG-ADC as a single dose of 0.3 MPK.

[0167] Figures 21A-21C show that the CD180-targeting DAR8 deruxtecan ADC CO-ADC-004 is highly active against disseminated primary AML cells in vivo. Figure 21A shows the characteristics of CTG-2240 primary AML. Figure 21B shows the results of in vivo analysis of hCD45 tumor (%) in bone marrow, monocytes in bone marrow, CD123 cells in bone marrow, and CD180+ LSC in bone marrow, CD180+ monocytes in bone marrow, CD180+ CD117+ in bone marrow, and CD180+ CD123+ in bone marrow after administration of 5MPK CO-ADC-004 compared to control (vehicle) and 5MPK IgG-ADC (CO-ADC-005).

[0168] FIG. 21C shows the results of in vivo analysis of %CD180+ LSC in bone marrow, %CD180+ monocytes in bone marrow, CD180+ CD117+ in bone marrow, and CD180+ CD123+ in bone marrow.

[0169] Example 8: Cellular Binding of CD180 ADCs to HEK293CD180 / MD-1 Cells and Potent Antitumor Efficacy In Vivo Anti-CD180 antibody CO-mAb-020 (also known as Ab-001 and CHAMPmAb-001) is a fully human IgG1, and its HCDR1, HCDR2, and HCDR3 (SEQ ID NOs: 1-3) and LCDR1, LCDR2, and LCDR3 (SEQ ID NOs: 4-6) are shown in Tables 1-2 above, respectively. The amino acid sequences of the VH, VL, HC, and LC of anti-CD180 antibody CO-mAb-020 are shown in Table 3 (SEQ ID NOs: 7, 8, and 11). The nucleotide sequences encoding the VH, VL, HC, and LC of anti-CD180 antibody CO-mAb-020 are shown in Table 4 (SEQ ID NOs: 9, 13, and 14).

[0170] The melting temperature, self-interaction, freeze / thaw stability, purity, multi-antigen reactivity, binding to CD180 / MD1 HEK293 cells, and internalization characteristics of the anti-CD180 antibody CO-mAb-020 in MV-4-11 are shown in Figures 22A-22G. The anti-CD180 antibody CO-mAb-20 (CO-ADC-004) DAR:8 (Figure 23B) or the anti-CD180 antibody CO-mAb-20 (CO-ADC-001) DAR:2.1 (Figure 23E) conjugated to deruxtecan or tesirin, respectively, were subjected to EC50 evaluation by cell-based ELISA using HEK293 cells expressing human D180 / MD-1. As shown in Figures 23G-23H, cell binding to HEK293 CD180 / MD-1 cells remained unchanged after conjugation.

[0171] The antitumor efficacy of the CO-ADC-004 lead ADC against MV4-11 tumors was tested in vivo. MV4-11 Luc tail vein injection (TVI) (utilizing a transvalvular insertion tool) was used for systemic engraftment of MV4-11 tumors. Mice were randomized once bioluminescence was detected above background in more than 90% of mice. A single dose of 5 mg / kg (MPK) was administered to each mouse via intravenous injection of either the therapeutic agent CO-ADC-004 or CO-ADC-005. Tumor burden was assessed by bioluminescence (Figure 24B). CO-ADC-004 administered at a dose of 5MPK demonstrated complete regression over a period of more than 30 days compared to the isotype-Dxd ADC CO-ADC-005 (Figure 24A). Thus, the ADC CO-ADC-004 is potent against MV4-11 tumors in vivo.

[0172] Example 9: Anti-CD180-Tesirin ADC effectively reduces tumor burden in vivo As described above, CO-ADC-001 is an anti-CD180 antibody-Tesirin ADC. CO-ADC-003 is an isotype-Tesirin ADC. The efficacy of CO-ADC-001 in reducing tumor burden was tested in vivo.

[0173] MV4-11 Luc tail vein injection (TVI) was used for systemic engraftment. Mice were randomized once bioluminescence was detected above background in more than 90% of mice. A single dose of 0.15 mg / kg (MPK) was administered to each mouse via intravenous injection of either the CO-ADC-001 ADC or CO-ADC-003 treatment. Tumor burden was assessed by bioluminescence (Figure 25B). CO-ADC-001 ADC administered at a dose of 0.15 MPK showed complete regression over a period of more than 30 days compared to the isotype-tesirin ADC CO-ADC-003 (Figure 25A).

[0174] Figures 26A-26C show that CD180 protein expression correlates with ADC cytotoxicity. Figures 26A-26B show that there is a wide range of sensitivity and cytotoxicity in AML patient samples. Figure 26C shows that CD180 protein expression correlates with IC 50 The correlation is shown.

[0175] Figures 27A-27C show the correlation between receptor expression and ADC efficacy in other comparative ADCs. Figure 27A shows CD19 expression correlating with in vitro activity of loncatecillin. Figure 27B shows CD33 expression correlating with in vitro activity of IMGN779. Figure 27C shows CD123 expression correlating with in vitro activity of SGN-CD123A.

[0176] While certain features of the invention have been specifically shown and described herein, many modifications, substitutions, variations, and equivalents will occur to those skilled in the art, and it is therefore to be understood that the appended claims are intended to cover all such modifications and variations as fall within the true spirit of the invention.

Claims

1. an anti-CD180 antibody drug conjugate (ADC) comprising a monoclonal antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors, a cytotoxic drug payload, and a linker moiety that conjugates the antibody or antigen-binding fragment thereof that targets CD180-highly expressing tumors to the cytotoxic drug payload, wherein the anti-CD180 antibody or antigen-binding fragment thereof comprises a set of three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a corresponding set of three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; A set of HCDR1, HCDR2 and HCDR3 and a corresponding set of LCDR1, LCDR2 and LCDR3 each have the amino acid sequence: HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1-3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4-6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1-3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5 and 15; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 45; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 54-56 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57-59; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 68-70 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 57, 58, and 71; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 80-82; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 91-93; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 102, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 103, and 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 112, 5, and 113; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4-6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4, 5, and 130; or HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24 and 25 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4 to 6; An anti-CD180 antibody drug conjugate (ADC) comprising:

2. The ADC of claim 1, wherein the anti-CD180 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region comprise the amino acid sequence of SEQ ID NOs: 7-8, 16-17, 27-28, 36-37, 46-47, 60-61, 72-73, 83-84, 94-95, 104-105, 114-115, 122-123, 131-132, or 139-140.

3. The ADC of claim 1, wherein the anti-CD180 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, and the heavy chain and light chain comprise the amino acid sequence of SEQ ID NOs: 11-12, 20-21, 31-32, 40-41, 50-51, 64-65, 76-77, 87-88, 98-99, 108-109, 118-119, 126-127, 135-136, or 143-144.

4. The anti-CD180 antibody or antigen-binding fragment thereof may be an IgG, Fv, scFv, Fab, F(ab') ２ 2. The ADC of claim 1, comprising a minibody, a diabody, a triabody, a nanobody, a bispecific antibody, a single domain antibody, or a chimeric antigen receptor.

5. 5. The ADC of claim 4, wherein the IgG is IgG1, IgG2, IgG3, or IgG4.

6. 2. The ADC of claim 1, wherein the cytotoxic drug payload comprises the ADC drug-linker conjugate tesirin (SG3249), wherein tesirin comprises the cytotoxic drug payload, wherein the cytotoxic drug payload comprises a pyrrolobenzodiazepine dimer cytotoxic DNA alkylating drug (SG3199), and wherein tesirin is conjugated to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-high expressing tumors via a cleavable linker moiety.

7. 2. The ADC of claim 1, comprising an ADC drug-linker conjugate deruxtecan, wherein the deruxetan comprises a cleavable linker, a self-immolative aminomethylene spacer, and a cytotoxic drug payload, wherein the cytotoxic drug payload comprises a topoisomerase 1 inhibitor payload, wherein the topoisomerase 1 inhibitor payload is a derivative of exatecan (DX-8951), and wherein the deruxetan is conjugated to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-high expressing tumors via a cleavable linker, wherein the cleavable linker is a glycine-glycine-phenylalanine-glycine tetrapeptide-based linker.

8. 2. The ADC of claim 1, wherein the cleavable linker moiety is a hydrazone linker, a disulfide linker, or a peptide linker.

9. 4. The ADC of claim 3, wherein the peptide linker is a dipeptide linker selected from the group consisting of valine-citrulline (Val-Cit), valine-alanine (Val-Ala), and alanine-alanine (Ala-Ala).

10. 5. The ADC of claim 4, wherein the dipeptide linker is linked to the cytotoxic drug payload by a spacer unit, and the spacer unit is para-aminobenzyloxycarbonyl (PABC).

11. 4. The ADC of claim 3, wherein the peptide linker is a tripeptide linker, wherein the tripeptide linker is a glutamic acid-valine-citrulline (EVCit) tripeptide linker.

12. 7. The ADC of claim 6, wherein a glutamic acid-valine-citrulline (EVCit) tripeptide linker is linked to the metamido-para-aminobenzylcarbamate (MA-PABC) group.

13. 7. The ADC of claim 6, wherein the drug-to-antibody ratio (DAR) is 2.

1.

14. 8. The ADC of claim 7, wherein the drug-to-antibody ratio (DAR) is 8.

15. A method for treating cancer in a subject, comprising administering to the subject the ADC of any one of claims 1 to 3 or 6 to 7.

16. 16. The method of claim 15, wherein the cancer is acute myeloid leukemia (AML), mantle cell lymphoma (MCL), multiple myeloma, follicular lymphoma, acute B-lymphoblastic leukemia, or diffuse large B-cell lymphoma.

17. 17. The method of claim 16, wherein the AML is primary AML or primary MCL.

18. 16. The method of claim 15, wherein the ADC comprises a set of HCDR1, HCDR2, and HCDR3 comprising the amino acids of SEQ ID NOs: 1-3 and a corresponding set of LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 4-6, and the cytotoxic drug payload comprises the pyrrolobenzodiazepine dimeric cytotoxic alkylating agent Tesirine (SG3199) conjugated to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-high expressing tumors via a cleavable linker moiety, wherein the cleavable linker moiety is a valine-alanine (Val-Ala) peptide linker.

19. 19. The method of claim 18, wherein the Val-Ala peptide linker is cleavable by cathepsin B.

20. 20. The method of claim 18, wherein the DAR is 2.

1.

21. 19. The method of claim 18, wherein the cancer is primary AML.

22. 19. The method of claim 18, wherein the cancer is primary MCL.

23. The ADC comprises a set of HCDR1, HCDR2 and HCDR3 comprising the amino acids of SEQ ID NOs: 1, 35 and 25 and a corresponding set of LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 5 and 26, and the ADC is conjugated to the ADC drug-linker conjugate deruxtecan, wherein deruxtetan is a cleavable linker based on glycine-glycine-phenylalanine-glycine, a self-immolative aminomethylene spirochete.

16. The method of claim 15, wherein the monoclonal antibody or antigen-binding fragment thereof is conjugated to a monoclonal antibody or antigen-binding fragment thereof that targets CD180-high expressing tumors via a cleavable glycine-glycine-phenylalanine-glycine tetrapeptide-based linker.

24. 24. The method of claim 23, wherein the DAR is 8.

25. 24. The method of claim 23, wherein the cancer is primary AML.

26. 24. The method of claim 23, wherein the cancer is primary MCL.

27. A method for modulating a CD180-mediated immune response in a subject, comprising administering to the subject the ADC of any one of claims 1 to 3 or 6 to 7.

28. 28. The method of claim 27, wherein the CD180-mediated immune response is antigen-presenting cell activity of B cells, antibody secretion, or proliferation of B lymphocytes, monocytes, or dendritic cells.

29. 28. The method of claim 27, wherein modulating the CD180-mediated immune response comprises enhancing the CD180-mediated immune response or inhibiting the CD180-mediated immune response.

30. A method for treating a disease in a subject, comprising administering to the subject the ADC of any one of claims 1 to 3 or 6 to 7.

31. 31. The method of claim 30, wherein the disease is a viral infection, a bacterial infection, an autoimmune disease or an immune disorder.

32. 1. An isolated anti-CD180 binding molecule comprising a set of three heavy chain complementarity determining regions, HCDR1, HCDR2 and HCDR3, and a corresponding set of three light chain complementarity determining regions, LCDR1, LCDR2 and LCDR3, A set of HCDR1, HCDR2 and HCDR3 and a corresponding set of LCDR1, LCDR2 and LCDR3 each have the amino acid sequence: HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1-3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 4-6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1-3 and SEQ ID NOs: 4, 5, and 15; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and SEQ ID NOs: 4, 5, and 26; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and SEQ ID NOs: 4, 5, and 45; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 54-56 and SEQ ID NOs: 57-59; SEQ ID NOs: 68-70 and HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 57, 58, and 71; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and SEQ ID NOs: 80-82; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 35, and 25 and SEQ ID NOs: 91-93; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 102, and 25 and SEQ ID NOs: 4, 103, and 6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 24, and 25 and SEQ ID NOs: 112, 5, and 113; SEQ ID NOs: 1, 35, and 25 and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 4-6; HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 1, 44, and 25 and SEQ ID NOs: 4, 5, and 130; SEQ ID NOs: 1, 24, and 25 and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 4 to 6; 1. An isolated anti-CD180 binding molecule comprising:

33. 33. The anti-CD180 binding molecule of claim 32, wherein the binding molecule comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequence of SEQ ID NOs:7-8, 16-17, 27-28, 36-37, 46-47, 60-61, 72-73, 83-84, 94-95, 104-105, 114-115, 122-123, 131-132, or 139-140.

34. 33. The anti-CD180 binding molecule of claim 32, wherein the binding molecule comprises a heavy chain and a light chain, and the heavy and light chains comprise the amino acid sequences of SEQ ID NOs: 11-12, 20-21, 31-32, 40-41, 50-51, 64-65, 76-77, 87-88, 98-99, 108-109, 118-119, 126-127, 135-136, or 143-144.

35. The binding molecule may be IgG, Fv, scFv, Fab, F(ab') ２ 33. The anti-CD180 binding molecule of claim 32, comprising a minibody, a diabody, a triabody, a nanobody, a bispecific antibody, a single domain antibody, or a chimeric antigen receptor.

36. 36. The anti-CD180 binding molecule of claim 35, wherein the IgG is IgG1, IgG2, IgG3, or IgG4.

37. An isolated polynucleotide sequence encoding the anti-CD180 binding molecule of any one of claims 32 to 36.

38. A vector comprising the polynucleotide sequence of claim 37.

39. 39. A host cell comprising the vector of claim 38.

40. A composition comprising the anti-CD180 binding molecule of any one of claims 32 to 36 and a pharmaceutically acceptable carrier.

41. A composition comprising the ADC of any one of claims 1 to 3 or 6 to 7 and a pharmaceutically acceptable carrier.

42. 38. A composition comprising the polynucleotide sequence of claim 37.

43. 43. The composition of claim 42, wherein the polynucleotide sequence comprises an expression vector for expressing the anti-CD180 binding molecule in a cell.

44. 42. A method of modulating a CD180-mediated immune response in a subject, comprising administering to the subject the composition of claim 40 or 41.

45. 45. The method of claim 44, wherein the CD180-mediated immune response is antigen-presenting cell activity of B cells, antibody secretion, or proliferation of B lymphocytes, monocytes, or dendritic cells.

46. The method of claim 44, wherein modulating a CD180-mediated immune response comprises enhancing a CD180-mediated immune response or inhibiting a CD180-mediated immune response.

47. 42. A method of treating a disease in a subject, comprising administering to the subject the composition of claim 40 or 41.

48. 48. The method of claim 47, wherein the disease is a viral infection, a bacterial infection, cancer, an autoimmune disease or an immune disorder.

49. 48. The method of claim 47, wherein the disease is a CD180-mediated disease.

50. 49. The method of claim 48, wherein the cancer is acute myeloid leukemia (AML), mantle cell lymphoma (MCL), multiple myeloma, follicular lymphoma, acute B-lymphoblastic leukemia, or diffuse large B-cell lymphoma.

51. A method for treating a cancer that highly expresses CD180 in a subject, comprising administering to the subject the composition of claim 41.

52. A method for treating a cancer that highly expresses CD180 in a subject, comprising administering to the subject a composition of the ADC of claim 6.

53. 53. The method of claim 52, wherein the cancer is acute myeloid leukemia (AML), mantle cell lymphoma (MCL), multiple myeloma, follicular lymphoma, acute B-lymphoblastic leukemia, or diffuse large B-cell lymphoma.

54. 53. The method of claim 52, wherein the cancer is primary AML.

55. 53. The method of claim 52, wherein the cancer is primary MCL.

56. A method for treating a cancer that highly expresses CD180 in a subject, comprising administering to the subject a composition of the ADC of claim 7.

57. 57. The method of claim 56, wherein the cancer is acute myeloid leukemia (AML), mantle cell lymphoma (MCL), multiple myeloma, follicular lymphoma, acute B-lymphoblastic leukemia, or diffuse large B-cell lymphoma.

58. 57. The method of claim 56, wherein the cancer is primary AML.

59. 57. The method of claim 56, wherein the cancer is primary MCL.

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