Anti-variable MUC1* antibodies and uses thereof

Abstract

Described herein are methods and compositions for targeted delivery of therapeutic agents and multispecific antibodies or antibody fragments that contain a binding domain for MUC1* and a binding domain for CD3. The disclosure also provides compositions comprising the antibodies, and methods for treating diseases and disorders, such as cancer.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 330,277, filed April 12, 2022, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on April 4, 2023, has the filename 56699-751.601_SL.xml and is 230 kilobytes in size.

[0003] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with disclosures contained herein, the specification is intended to supersede and / or take precedence over such conflicting material. [Background technology]

[0004] Provided herein are multispecific antibodies comprising a MUC1* binding domain and a CD3 binding domain. Also provided herein are antibody conjugates of the formulations described herein that comprise one or more moieties derived from a therapeutic agent (e.g., a topoisomerase I inhibitor, a tubulin formation inhibitor), further comprising a polypeptide, such as an antibody, that binds to a target of interest (e.g., an antibody that targets MUC1*). The multispecific antibodies and antibody-drug conjugates described above are useful for treating diseases or disorders, e.g., proliferative diseases such as cancer. Also provided herein are uses and methods for treating diseases and disorders using these multispecific antibodies and antibody conjugates. Summary of the Invention

[0005] As used herein, the compound of formula (I):

[0006] [ka] wherein X is a compound capable of inhibiting topoisomerase I or a moiety derived from a compound capable of inhibiting tubulin formation, R is a binding moiety, L is a dipeptide or tripeptide or tetrapeptide binding moiety having Z attached to the N-terminus and R attached to the C-terminus, and [Ab] is an antibody comprising an anti-MUC1* binding domain comprising three heavy chain (HC) complementarity determining regions (CDRs): MUC1* HC-CDR1, MUC1* HC-CDR2, and MUC1* HC-CDR3, wherein MUC1* HC-CDR1, MUC1* HC-CDR2, and MUC1* HC-CDR3 of the MUC1* binding domain comprise an amino acid sequence selected from those set out in Table 1, and the MUC1* binding domain comprises three light chain (LC) complementarity determining regions (CDRs): MUC1* LC-CDR1, MUC1* LC-CDR2, and MUC1* and y is an integer from 1 to 10.

[0007] L may include valine and citrulline. L may include glycine and phenylalanine. R may include para-aminobenzyl. R may include

[0008] [ka] where * indicates the point of attachment of the X group. R can include a moiety containing the structure:

[0009] [ka] where * indicates the point of attachment of the X group. R may include a moiety comprising

[0010] [ka] where * indicates the point of attachment of the X group.

[0011] L is

[0012] [ka] L can be a dipeptide linking moiety comprising the structure

[0013] [ka] The linking moiety may be a tetrapeptide linking moiety comprising the structure:

[0014] X can be MMAE or MMAF. X can be exatecan or Dxd. R can be

[0015] [ka] where * indicates the point of attachment of the X group, and X is Dxd. R can include a moiety containing the structure:

[0016] [ka] where * indicates the point of attachment of the X group, and X is exatecan.

[0017] The antibody may be of isotype IgG1 or IgG2. The antibody may be of isotype IgG2b.

[0018] An anti-MUC1* binding domain may comprise a heavy chain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 38 or 44. An anti-MUC1* binding domain may comprise a heavy chain variable domain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 39 or 45.

[0019] An anti-MUC1* binding domain may comprise a light chain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 41 or 47. An anti-MUC1* binding domain may comprise a light chain variable domain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 42 or 48.

[0020] The anti-MUC1* binding domain may comprise a single chain variable fragment (scFv) comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 129 or 130.

[0021] Disclosed herein is an antibody conjugate comprising an antibody comprising an anti-MUC1* binding domain, the antibody being conjugated to a payload via a maleimide-cysteine ​​bond, the payload comprising a linker and a cytotoxic compound, the cytotoxic compound comprising a tubulin inhibitor or a topoisomerase I inhibitor. The linker may comprise valine. The linker may comprise citrulline. The linker may comprise valine and citrulline. The linker may comprise

[0022] [ka] The linker may be a dipeptide linking moiety comprising the structure: The linker may comprise at least one glycine. The linker may comprise at least one glycine and a phenylalanine. The linker may be a dipeptide linking moiety comprising the structure:

[0023] [ka] The linker may include a structure of: The linker may include para-aminobenzyl. The linker may include a structure of:

[0024] [ka] where * indicates the point of attachment of the cytotoxic group.

[0025] [ka] where * indicates the point of attachment of the cytotoxic group. The tubulin inhibitor can be MMAE or MMAF. The topoisomerase I inhibitor can be a derivative of exatecan or deruxtecan. The linker can be

[0026] [ka] where * indicates the point of attachment of the cytotoxic group and the topoisomerase I inhibitor is Dxd.

[0027] [ka] where * indicates the point of attachment of the cytotoxic group and the topoisomerase I inhibitor is exatecan.

[0028] The antibody conjugate may have the structure provided below:

[0029] [ka] where n is 1 to 10.

[0030] The antibody conjugate may have the structure provided below:

[0031] [ka] where n is 1 to 10.

[0032] The antibody isotype may be IgG1 or IgG2. The antibody isotype may be IgG2. The antibody may be conjugated to at least two payloads. The antibody may be conjugated to at least three payloads. The antibody may be conjugated to at least four payloads. The antibody may be conjugated to at least five payloads. The antibody may be conjugated to at least six payloads. The antibody may be conjugated to at least seven payloads. The antibody may be conjugated to at least eight payloads. The anti-MUC1* binding domain may comprise three light chain (LC) complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, and the LC-CDR1, LC-CDR2, and LC-CDR3 of the MUC1* binding domain may comprise an amino acid sequence selected from those shown in Table 1, and at least one of the LC-CDR1, LC-CDR2, and LC-CDR3 may comprise 0-2 amino acid modifications. The anti-MUC1* binding domain may comprise three heavy chain (HC) complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, where the HC-CDR1, HC-CDR2, and HC-CDR3 of the MUC1* binding domain may comprise an amino acid sequence selected from those shown in Table 1, and where at least one of the HC-CDR1, HC-CDR2, and HC-CDR3 may comprise 0-2 amino acid modifications. The anti-MUC1* binding domain may comprise a heavy chain variable domain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence shown in Table 2. An anti-MUC1* binding domain may comprise a light chain variable domain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence shown in Table 2.The anti-MUC1* binding domain may comprise a single chain variable fragment (scFv) comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence shown in Table 3.

[0033] Provided herein is an antibody comprising a MUC1* binding domain and a CD3 binding domain, wherein the anti-MUC1* binding domain may comprise three heavy chain (HC) complementarity determining regions (CDRs): MUC1* HC-CDR1, MUC1* HC-CDR2, and MUC1* HC-CDR3, wherein MUC1* HC-CDR1 may comprise the amino acid sequence of SEQ ID NO: 1, wherein MUC1* HC-CDR2 may comprise the amino acid sequence of SEQ ID NO: 2, and wherein MUC1* HC-CDR3 may comprise the amino acid sequence of SEQ ID NO: 3, wherein the MUC1* binding domain may comprise three light chain (LC) complementarity determining regions (CDRs): MUC1* LC-CDR1, MUC1* LC-CDR2, and MUC1* LC-CDR3, wherein MUC1* LC-CDR1 may comprise the amino acid sequence of SEQ ID NO: 13, and wherein MUC1* LC-CDR2 may comprise the amino acid sequence of SEQ ID NO: 14, and CD3 HC-CDR3, an amino acid sequence of SEQ ID NO: 15; and the CD3 binding domain may comprise three heavy chain (HC) complementarity determining regions (CDRs): CD3 HC-CDR1, CD3 HC-CDR2, and CD3 HC-CDR3, and the CD3 HC-CDR1, CD3 HC-CDR2, and CD3 HC-CDR3 of the CD3 binding domain may comprise an amino acid sequence selected from those shown in Table 2; and the CD3 binding domain may comprise three light chain (LC) complementarity determining regions (CDRs): CD3 LC-CDR1, CD3 LC-CDR2, and CD3 LC-CDR3, and the CD3 LC-CDR1, CD3 LC-CDR2, and CD3 LC-CDR3 of the CD3 binding domain may comprise an amino acid sequence selected from those shown in Table 4.

[0034] The antibody may comprise an Fc domain. The Fc domain may be a heterodimeric Fc domain. The heterodimeric Fc domain may comprise a knob chain and a hole chain to form a knob-in-hole (KiH) structure. The knob chain may comprise a sequence having at least about 95% identity to a sequence selected from SEQ ID NO: 121, 122, 123, or 124. The hole chain may comprise a sequence having at least about 95% identity to a sequence selected from SEQ ID NO: 125, 126, 127, or 128. The MUC1* binding domain may comprise a heavy chain variable domain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from Table 2. The MUC1* binding domain may comprise a light chain variable domain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from Table 2. The MUC1* binding domain may comprise a single chain variable fragment (scFv) comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from Table 3. The CD3 binding domain may comprise a heavy chain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 26 or 31. The CD3 binding domain may comprise a light chain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 29 or 35.The CD3 binding domain may comprise a single chain variable fragment (scFv) comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 131 or 132. The antibody may comprise a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 50, 52, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, or 114.

[0035] Provided herein is a method of treating cancer, comprising administering to a subject in need thereof an antibody according to any one of claims 1 to 64. In some embodiments, the cancer expresses MUC1*. The cancer may be breast cancer, colon cancer, prostate cancer, pancreatic cancer, or lung cancer. [Brief description of the drawings]

[0036] [Figure 1-1]Figure 1 shows photographs of T47D, MUC1*-positive breast cancer cells cultured with human T cells supplemented with various concentrations of the bispecific antibody 20A10-OKT3-BiTE. 20A10 is a humanized anti-MUC1* antibody, and OKT3 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates the binding of T cells to the cancer cells, shown in the figure as clustering of T cells, which is a sign of activation induced by the bispecific bridge between T cell CD3 and cancer cell MUC1*. In Figure 1A, the concentration of the bispecific antibody is 1,000 ng / mL. In Figure 1B, the concentration is 333 ng / mL. In Figure 1C, the concentration is 111 ng / mL. In Figure 1D, the concentration is 37 ng / mL. In Figure 1E, the concentration is 12.3 ng / mL. In Figure 1F, the concentration is 4.1 ng / mL. In Figure 1G, the concentration is 1.3 ng / mL. In Figure 1H, the concentration is 0.4 ng / mL. [Figure 1-2] Figure 1 shows photographs of T47D, MUC1* positive breast cancer cells cultured with human T cells with various concentrations of bispecific antibody 20A10-OKT3-BiTE. 20A10 is a humanized anti-MUC1* antibody and OKT3 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates the binding of T cells to cancer cells, shown in the figure as clustering of T cells, which is a sign of activation induced by the bispecific bridge between T cell CD3 and cancer cell MUC1*. In Figure 1I, the concentration is 0.15 ng / mL. In Figure 1J, the concentration is 0.05 ng / mL. Figure 1K is a control well in which both T cells and cancer cells are present but no bispecific antibody is added. Figure 1L is a control well in which only cancer cells are present. [Figure 1-3]Figure 1 shows photographs of T47D, MUC1* positive breast cancer cells cultured with human T cells supplemented with various concentrations of the bispecific antibody 20A10-OKT3-BiTE. 20A10 is a humanized anti-MUC1* antibody, and OKT3 is an antibody that binds to CD3 present on human T cells. As can be seen, addition of the bispecific antibody mediates binding of the T cells to the cancer cells, shown in the figure as clustering of the T cells, a sign of activation induced by the bispecific bridge between T cell CD3 and cancer cell MUC1*. Figure 1M shows a cartoon depicting how the LDH cell cytotoxicity assay works, with higher readings at A490 indicating more effective cell killing. Figure 1N shows a graph of cell killing as a function of concentration of 20A10-OKT3, an anti-MUC1* / anti-CD3 bispecific antibody. [Figure 1-4] Figure 10 shows a photograph of T47D, MUC1* positive breast cancer cells cultured with human T cells loaded with various concentrations of bispecific antibody 20A10-OKT3-BiTE. 20A10 is a humanized anti-MUC1* antibody and OKT3 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates the binding of the T cells to the cancer cells, shown in the figure as clustering of the T cells, which is a sign of activation induced by the bispecific bridge between T cell CD3 and cancer cell MUC1*. Figure 10 shows a graph of secreted interferon gamma secreted by the T cells as a function of added anti-MUC1* / anti-CD3 bispecific antibody, where the anti-MUC1* antibody is 20A10 and the anti-CD3 antibody is OKT3. [Figure 1-5]Figure 1B shows a photograph of T47D, MUC1* positive breast cancer cells cultured with human T cells loaded with various concentrations of the bispecific antibody 20A10-OKT3-BiTE. 20A10 is a humanized anti-MUC1* antibody and OKT3 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates the binding of the T cells to the cancer cells, shown in the figure as clustering of the T cells, which is a sign of activation induced by the bispecific bridge between T cell CD3 and cancer cell MUC1*. Figure 1P shows a graph of secreted TNFα secreted by T cells as a function of added anti-MUC1* / anti-CD3 bispecific antibody, where the anti-MUC1* antibody is 20A10 and the anti-CD3 antibody is OKT3. [Figure 2-1] Figure 2 shows photographs of T47D, MUC1* positive breast cancer cells cultured with human T cells supplemented with various concentrations of bispecific antibody 20A10-12F6-BiTE. 20A10 is a humanized anti-MUC1* antibody and 12F6 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates the binding of T cells to the cancer cells, shown in the figure as clustering of T cells, which is a sign of activation induced by the bispecific bridge between T cell CD3 and cancer cell MUC1*. In Figure 2A, the concentration of the bispecific antibody is 1,000 ng / mL. In Figure 2B, the concentration is 333 ng / mL. In Figure 2C, the concentration is 111 ng / mL. In Figure 2D, the concentration is 37 ng / mL. In Figure 2E, the concentration is 12.3 ng / mL. In Figure 2F, the concentration is 4.1 ng / mL. In Figure 2G, the concentration is 1.3 ng / mL. In Figure 2H, the concentration is 0.4 ng / mL. [Figure 2-2]Figure 2 shows photographs of T47D, MUC1* positive breast cancer cells cultured with human T cells with various concentrations of bispecific antibody 20A10-12F6-BiTE. 20A10 is a humanized anti-MUC1* antibody and 12F6 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates the binding of T cells to cancer cells, shown in the figure as clustering of T cells, which is a sign of activation induced by the bispecific bridge between T cell CD3 and cancer cell MUC1*. In Figure 2I, the concentration is 0.15 ng / mL. In Figure 2J, the concentration is 0.05 ng / mL. Figure 2K is a control well in which both T cells and cancer cells are present but no bispecific antibody is added. Figure 2L is a control well in which only cancer cells are present. [Figure 2-3] Figure 2 shows photographs of T47D, MUC1* positive breast cancer cells cultured with human T cells supplemented with various concentrations of the bispecific antibody 20A10-12F6-BiTE. 20A10 is a humanized anti-MUC1* antibody and 12F6 is an antibody that binds to CD3 present on human T cells. As can be seen, addition of the bispecific antibody mediates binding of the T cells to the cancer cells, shown in the figure as clustering of the T cells, a sign of activation induced by the bispecific bridge between T cell CD3 and cancer cell MUC1*. Figure 2M shows a cartoon depicting how the LDH cytotoxicity assay works, with higher readings at A490 indicating more effective cell killing. Figure 2N shows a graph of cell killing as a function of concentration of 20A10-OKT3, an anti-MUC1* / anti-CD3 bispecific antibody. [Figure 2-4]Figure 2 shows a photograph of T47D, MUC1* positive breast cancer cells cultured with human T cells loaded with various concentrations of bispecific antibody 20A10-12F6-BiTE. 20A10 is a humanized anti-MUC1* antibody and 12F6 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates the binding of T cells to the cancer cells, shown in the figure as clustering of T cells, which is a sign of activation induced by the bispecific bridge between T cell CD3 and cancer cell MUC1*. Figure 2O shows a graph of secreted interferon gamma secreted by T cells as a function of added anti-MUC1* / anti-CD3 bispecific antibody, where the anti-MUC1* antibody is 20A10 and the anti-CD3 antibody is 12F6. [Figure 2-5] Figure 2B shows a photograph of T47D, MUC1* positive breast cancer cells cultured with human T cells loaded with various concentrations of the bispecific antibody 20A10-12F6-BiTE. 20A10 is a humanized anti-MUC1* antibody and 12F6 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates the binding of the T cells to the cancer cells, shown in the figure as clustering of the T cells, which is an indication of activation induced by the bispecific bridge between T cell CD3 and cancer cell MUC1*. Figure 2P shows a graph of secreted TNFα secreted by the T cells as a function of added anti-MUC1* / anti-CD3 bispecific antibody, where the anti-MUC1* antibody is 20A10 and the anti-CD3 antibody is 12F6. [Figure 3-1]Figure 3 shows photographs of HCT-MUC1* transduced cancer cells cultured with human T cells supplemented with various concentrations of bispecific antibody 20A10-OKT3-BiTE. 20A10 is a humanized anti-MUC1* antibody and OKT3 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates binding of the T cells to the cancer cells, shown in the figure as clustering of the cells. In Figure 3A, the concentration of the bispecific antibody is 1,000 ng / mL. In Figure 3B, the concentration is 333 ng / mL. In Figure 3C, the concentration is 111 ng / mL. In Figure 3D, the concentration is 37 ng / mL. In Figure 3E, the concentration is 12.3 ng / mL. In Figure 3F, the concentration is 4.1 ng / mL. In Figure 3G, the concentration is 1.3 ng / mL. In Figure 3H, the concentration is 0.4 ng / mL. [Figure 3-2] Figure 3 shows photographs of HCT-MUC1* transduced cancer cells cultured with human T cells with various concentrations of bispecific antibody 20A10-OKT3-BiTE. 20A10 is a humanized anti-MUC1* antibody and OKT3 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates binding of the T cells to the cancer cells, shown in the figure as clustering of the cells. In Figure 3I, the concentration is 0.15 ng / mL. In Figure 3J, the concentration is 0.05 ng / mL. Figure 3K is a control well in which both T cells and cancer cells are present but no bispecific antibody is added. Figure 3L is a control well in which only cancer cells are present. [Figure 4-1]Photographs of HCT-MUC1* transduced cancer cells cultured with human T cells supplemented with various concentrations of the bispecific antibody 20A10-12F6-BiTE. 20A10 is a humanized anti-MUC1* antibody, and 12F6 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates binding of the T cells to the cancer cells, shown in the figure as clustering of the cells. In FIG. 4A, the concentration of the bispecific antibody is 1,000 ng / mL. In FIG. 4B, the concentration is 333 ng / mL. In FIG. 4C, the concentration is 111 ng / mL. In FIG. 4D, the concentration is 37 ng / mL. In FIG. 4E, the concentration is 12.3 ng / mL. In FIG. 4F, the concentration is 4.1 ng / mL. In FIG. 4G, the concentration is 1.3 ng / mL. In FIG. 4H, the concentration is 0.4 ng / mL. [Figure 4-2] Photographs of HCT-MUC1* transduced cancer cells cultured with human T cells with various concentrations of bispecific antibody 20A10-12F6-BiTE added. 20A10 is a humanized anti-MUC1* antibody and 12F6 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates binding of the T cells to the cancer cells, shown in the figure as clustering of the cells. In FIG. 4I, the concentration is 0.15 ng / mL. In FIG. 4J, the concentration is 0.05 ng / mL. FIG. 4K is a control well in which both T cells and cancer cells are present but no bispecific antibody is added. FIG. 4L is a control well in which the bispecific antibody is added to the cancer cells but no T cells are present. [Diagram 5]Figure 5 shows photographs of HCT-WT cancer cells that are negative for MUC1 and MUC1*. Cancer cells were incubated with MMNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently attached to deglycosylated MNC2, which binds MUC1*. As can be seen, the addition of MNC2-ADC did not affect the viability of these HCT, MUC1-negative cells. In Figure 5A, the concentration of MNC2-ADC is 100 nM. In Figure 5B, the concentration of MNC2-ADC is 10 nM. In Figure 5C, the concentration of MNC2-ADC is 0.1 nM. In Figure 5D, the concentration of MNC2-ADC is 0.01 nM. In Figure 5E, the concentration of MNC2-ADC is 0.001 nM. In Figure 5F, the concentration of MNC2-ADC is 0 nM. [Figure 6] Figure 6 shows photographs of HCT-MUC1* cancer cells. These HCT cells were transduced to express MUC1*, the target of the antibody MNC2. The cancer cells were incubated with MMNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently attached to deglycosylated MNC2, which binds MUC1*. As can be seen, the addition of MNC2-ADC at 100 nM, the highest concentration tested here, induced cell clumping, an indicator of cell death. In Figure 6A, the concentration of MNC2-ADC is 100 nM. In Figure 6B, the concentration of MNC2-ADC is 10 nM. In Figure 6C, the concentration of MNC2-ADC is 1.0 nM. In Figure 6D, the concentration of MNC2-ADC is 0.1 nM. In Figure 6E, the concentration of MNC2-ADC is 0.01 nM. In Figure 6F, the concentration of MNC2-ADC is 0.001 nM. In Figure 6G, the concentration of MNC2-ADC is 0 nM. [Figure 7]Photographs of K562-WT cancer cells that are negative for MUC1 and MUC1* are shown. The cancer cells were incubated with MMNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently attached to deglycosylated MNC2, which binds MUC1*. As can be seen in the figure, the addition of MNC2-ADC did not affect the viability of these MUC1-negative cells. In FIG. 7A, the concentration of MNC2-ADC is 100 nM. In FIG. 7B, the concentration of MNC2-ADC is 10 nM. In FIG. 7C, the concentration of MNC2-ADC is 1.0 nM. In FIG. 7D, the concentration of MNC2-ADC is 0.1 nM. In FIG. 7E, the concentration of MNC2-ADC is 0.01 nM. In FIG. 7F, the concentration of MNC2-ADC is 0.001. In Figure 7G, the concentration of MNC2-ADC is 0 nM. [Figure 8] 8A shows a photograph of K562-MUC1* cells. These MUC1*-negative cells were transduced to express MUC1*. The cancer cells were incubated with MMNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently attached to deglycosylated MNC2, which binds MUC1*. As can be seen, the addition of MNC2-ADC at the higher concentrations tested here, 10 nM and 100 nM, induced cell clumping, an indicator of cell death. In FIG. 8A, the concentration of MNC2-ADC is 100 nM. In FIG. 8B, the concentration of MNC2-ADC is 10 nM. In FIG. 8C, the concentration of MNC2-ADC is 1.0 nM. In FIG. 8D, the concentration of MNC2-ADC is 0.1 nM. In FIG. 8E, the concentration of MNC2-ADC is 0.01 nM. In Figure 8F, the concentration of MNC2-ADC is 0.001 nM. In Figure 8G, the concentration of MNC2-ADC is 0 nM. [Figure 9A]FIG. 13 shows a graph of a cell viability assay, where PrestoBlue was used to measure cell death. The graph shows cell viability as a function of MNC2-ADC concentration, comparing the effect of MNC2-ADC on K562-WT cells to the effect on K562-MUC1* cells. As can be seen, the MNC2-ADC antibody only induced death of MUC1* expressing cells at concentrations of 10 nM and 100 nM. [Figure 9B] FIG. 9B shows a graph of a cell viability assay, using PrestoBlue to measure cell death. FIG. 9B shows cell viability as a function of MNC2-ADC concentration, comparing the effect of MNC2-ADC on HCT-WT cells to the effect on HCT-MUC1* cells. As can be seen, the MNC2-ADC antibody only induced death of MUC1* expressing cells at high concentrations. [Figure 10] Figure 10 shows photographs of T47D-wt breast cancer cells expressing both full-length MUC1, which MNC2 does not bind, and MUC1*, which MNC2 binds. Cancer cells were incubated with MMNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently attached to deglycosylated MNC2, which binds MUC1*. In Figure 10A, the concentration of MNC2-ADC is 100 nM. In Figure 10B, the concentration of MNC2-ADC is 10 nM. In Figure 10C, the concentration of MNC2-ADC is 1.0 nM. In Figure 10D, the concentration of MNC2-ADC is 0.1 nM. In Figure 10E, the concentration of MNC2-ADC is 0.01 nM. In Figure 10F, the concentration of MNC2-ADC is 0.001. In Figure 10G, the concentration of MNC2-ADC is 0 nM. [Figure 11]Figure 11 shows a photograph of T47D-MUC1* cells transduced to express more MUC1*. Cancer cells were incubated with MMNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently attached to deglycosylated MNC2, which binds MUC1*. As can be seen, the addition of MNC2-ADC at the higher concentrations tested here, 10 nM and 100 nM, induced cell clumping, an indicator of cell death. In Figure 11A, the concentration of MNC2-ADC is 100 nM. In Figure 11B, the concentration of MNC2-ADC is 10 nM. In Figure 11C, the concentration of MNC2-ADC is 1.0 nM. In Figure 11D, the concentration of MNC2-ADC is 0.1 nM. In Figure 11E, the concentration of MNC2-ADC is 0.01 nM. In Figure 11F, the concentration of MNC2-ADC is 0.001. In Figure 11G, the concentration of MNC2-ADC is 0 nM. [Figure 12] Figure 1 shows a graph of a cell viability assay, using PrestoBlue to detect dead cells. The graph shows cell viability as a function of MNC2-ADC concentration, comparing the effect of MNC2-ADC on T47D-WT cells to the effect on T47D-MUC1* cells. As can be seen, the MNC2-ADC antibody induced death of MUC1* expressing cells at concentrations of 10 nM and 100 nM. [Figure 13]Figure 13 shows a photograph of T47D-wt breast cancer cells expressing both full-length MUC1, which MNC2 does not bind, and MUC1*, which MNC2 binds. Cancer cells were incubated with MMNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently attached to deglycosylated MNC2, which binds MUC1*. As can be seen, MNC2-ADC antibody induced the death of T47D-WT cells at the highest concentration of MNC2-ADC, 1000 nM. In Figure 13A, the concentration of MNC2-ADC is 0 nM. In Figure 13B, the concentration of MNC2-ADC is 0.1 nM. In Figure 13C, the concentration of MNC2-ADC is 0.39 nM. In Figure 13D, the concentration of MNC2-ADC is 1.0 nM. In Figure 13E, the concentration of MNC2-ADC is 3.9 nM. In Figure 13F, the concentration of MNC2-ADC is 10 nM. In Figure 13G, the concentration of MNC2-ADC is 39 nM. In Figure 13H, the concentration of MNC2-ADC is 100 nM. In Figure 13I, the concentration of MNC2-ADC is 393 nM. In Figure 13J, the concentration of MNC2-ADC is 1000 nM. [Figure 14]Figure 14 shows a photograph of T47D-MUC1* cells transduced to express more MUC1*. Cancer cells were incubated with MMNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently attached to deglycosylated MNC2, which binds MUC1*. As can be seen, the addition of MNC2-ADC at the higher concentrations tested here, 10nM, 39nM, 100nM, 393nM, and 1000nM, induced cell clumping, an indicator of cell death. In Figure 14A, the concentration of MNC2-ADC is 0nM. In Figure 14B, the concentration of MNC2-ADC is 0.1nM. In Figure 14C, the concentration of MNC2-ADC is 0.39nM. In Figure 14D, the concentration of MNC2-ADC is 1.0nM. In Figure 14E, the concentration of MNC2-ADC is 3.9 nM. In Figure 14F, the concentration of MNC2-ADC is 10 nM. In Figure 14G, the concentration of MNC2-ADC is 39 nM. In Figure 14H, the concentration of MNC2-ADC is 100 nM. In Figure 14I, the concentration of MNC2-ADC is 393 nM. In Figure 14J, the concentration of MNC2-ADC is 1000 nM. [Figure 15] FIG. 13 shows a graph of a cell viability assay, where PrestoBlue was used to measure cell death. The graph shows cell viability as a function of MNC2-ADC concentration, comparing the effect of MNC2-ADC on T47D-WT cells to the effect on T47D-MUC1* cells. As can be seen, the MNC2-ADC antibody induced death of T47D-WT cells at the highest concentration of 1000 nM MNC2-ADC, while T47D-MUC1* cells were killed at concentrations of 10 nM, 39 nM, 100 nM, 393 nM, and 1000 nM. [Figure 16]16A shows a magnified view of cancer cells with various concentrations of MNC2-ADC added. Here, the toxin conjugated to the antibody is MMAE. FIG. 16A shows a photograph of breast cancer cells T47D wild type with MNC2-ADC added at concentrations ranging from 500 ng / mL to 0.1 ng / mL. As a control, no MNC2-ADC was added. The photograph was taken 72 hours after MNC2-ADC was added to the cancer cells. FIG. 16B shows a photograph of breast cancer cells T47D-MUC1* with MNC2-ADC added at concentrations ranging from 500 ng / mL to 0.1 ng / mL, showing that the cells are stably transfected with more MUC1* than naturally expressed. As a control, no MNC2-ADC was added. The photograph was taken 72 hours after MNC2-ADC was added to the cancer cells. FIG. 16C shows a photograph of breast cancer cells T47D wild type to which MNC2-ADC was added at concentrations ranging from 500 ng / mL to 0.1 ng / mL. As a control, no MNC2-ADC was added. In this case, MNC2-ADC was removed after 16 hours and the medium was replaced. The photograph was taken 72 hours after MNC2-ADC was initially added to the cancer cells. FIG. 16D shows a photograph of breast cancer cells T47D-MUC1*, showing that the cells are stably transfected with more MUC1* than they naturally express, to which MNC2-ADC was added at concentrations ranging from 500 ng / mL to 0.1 ng / mL. As a control, no MNC2-ADC was added. In this case, MNC2-ADC was removed after 16 hours and the medium was replaced. The photograph was taken 72 hours after MNC2-ADC was initially added to the cancer cells. Figure 16E shows a magnified image of T47D-wt cells treated with 1 μM taxol for 72 hours, and Figure 16F shows a magnified image of T47D-MUC1* cells treated with 1 μM taxol for 72 hours. [Figure 17]Figure 1 shows a graph of cancer cell death as a function of the concentration of MNC2-ADC MMAE added to cell culture medium for either 72 hours or 16 hours. In the latter case, after 16 hours, the medium is replaced with medium without MNC2-ADC and the experiment is continued until 72 hours after the initial addition of MNC2-ADC to the cancer cells. Cell viability is measured using Presto Blue. Cell death is normalized to taxol added to a final concentration of 1 μM, and the percent viable cells at 72 hours is normalized to 0% viability. [Figure 18]18A shows a magnified photograph of cancer cells with various concentrations of 20A10-ADC added. Here, the toxin conjugated to the antibody is MMAE. FIG. 18A shows a photograph of breast cancer cells T47D wild type with 20A10-ADC added at concentrations ranging from 500 ng / mL to 0.1 ng / mL. As a control, no 20A10-ADC was added. Photographs were taken 72 hours after 20A10-ADC was added to the cancer cells. FIG. 18B shows a photograph of breast cancer cells T47D-MUC1* with 20A10-ADC added at concentrations ranging from 500 ng / mL to 0.1 ng / mL, showing that the cells are stably transfected with more MUC1* than they naturally express. As a control, no 20A10-ADC was added. Photographs were taken 72 hours after 20A10-ADC was added to the cancer cells. FIG. 18C shows a photograph of breast cancer cells T47D wild type to which 20A10-ADC was added at concentrations ranging from 500 ng / mL to 0.1 ng / mL. As a control, no 20A10-ADC was added. In this case, 20A10-ADC was removed after 16 hours and the medium was replaced. The photograph was taken 72 hours after 20A10-ADC was initially added to the cancer cells. FIG. 18D shows a photograph of breast cancer cells T47D-MUC1*, showing that the cells are stably transfected with more MUC1* than they naturally express, to which 20A10-ADC was added at concentrations ranging from 500 ng / mL to 0.1 ng / mL. As a control, no 20A10-ADC was added. In this case, 20A10-ADC was removed after 16 hours and the medium was replaced. The photograph was taken 72 hours after 20A10-ADC was initially added to the cancer cells. Figure 18E shows a magnified image of T47D-wt cells treated with 1 μM taxol for 72 hours, and Figure 18F shows a magnified image of T47D-MUC1* cells treated with 1 μM taxol for 72 hours. [Figure 19]Figure 1 shows a graph of cancer cell death as a function of concentration of 20A10-ADC MMAE added to cell culture media for either 72 hours or 16 hours. In the latter case, after 16 hours, the media is replaced with media without 20A10-ADC and the experiment is allowed to continue until 72 hours after initial addition of 20A10-ADC to the cancer cells. Cell viability is measured using Presto Blue. Cell death is normalized to taxol added to a final concentration of 1 μM, and percent viable cells at 72 hours are normalized to 0% viability. [Figure 20] Figure 20A shows a graph of the fitting data for measuring IC50 and the data in tabular form. Figure 20A shows a graph of the IC50 fitting data of cancer cell killing mediated by either MNC2-ADC or 20A10-ADC, where the cancer cells are either T47D-wt or T47D-MUC1*, and the ADC is incubated with the target cancer cells for either 16 hours or 72 hours. Figure 20B shows a table listing the IC50 of each ADC for each cell type and incubation condition. [Figure 21] MN20A10-OKT3 knobs in whole format. FIG. 21A shows a photograph of T47D, MUC1* positive breast cancer cells cultured with human T cells loaded with various concentrations of bispecific antibody 20A10-OKT3-BiTE. 20A10 is a humanized anti-MUC1* antibody and OKT3 is an antibody that binds to CD3 present on human T cells. As can be seen, the addition of the bispecific antibody mediates the binding of T cells to the cancer cells, shown in the figure as clustering of T cells, which is a sign of activation induced by the bispecific bridge between T cell CD3 and cancer cell MUC1*. FIG. 21B shows a graph of secreted interferon gamma secreted by T cells as a function of added anti-MUC1* / anti-CD3 bispecific antibody, where the anti-MUC1* antibody is 20A10 and the anti-CD3 antibody is OKT3. FIG. 21C shows a graph of cell killing as a function of concentration of 20A10-OKT3, an anti-MUC1* / anti-CD3 bispecific antibody. [Figure 22] 22A-12F6 in various bispecific formats. The figure shows the killing curves of T47D-wt or T47D-MUC1* by bispecific antibodies in different formats. FIG. 22B shows a graph of T cell-mediated cancer cell killing in the presence of 12F6-MN20A10 bispecific. FIG. 22A is a cartoon of the 12F6-MN20A10N bispecific format used in FIG. 22B. FIG. 22D shows a graph of T cell-mediated cancer cell killing in the presence of MN20A10-12F6 KiH bispecific. FIG. 22C is a cartoon of the KiH bispecific format used in FIG. 22D. FIG. 22F shows a graph of T cell-mediated cancer cell killing in the presence of OKT3-MN20A10 bispecific. FIG. 22E is a cartoon of the OKT3-MN20A10 bispecific format used in FIG. 22F. Figure 22H shows a graph of T cell mediated cancer cell killing in the presence of MN20A10-12F6 chemically linked bispecific. Figure 22G is a cartoon of the MN20A10-12F6 bispecific format used in Figure 22H. [Figure 23] FIG. 1 shows the IC50 of MN20A10-OKT3 or MN20A10-12F6 in various bispecific formats. [Figure 24] FIG. 1 shows the IC50 of MN20A10-OKT3 or MN20A10-12F6 in various bispecific formats. [Diagram 25] Figure 25 shows a magnified image of human cancer cells stained with anti-MUC1* antibody MNC2, where the nuclei of the cells are stained blue with DAPI and the MNC2 antibody fluoresces red. Figure 25A shows that at time zero, the antibody is bound to the surface of the cells. Figure 25B shows that after 45 minutes, the antibody has been internalized and can be found throughout the cytoplasm. Antibody internalization is necessary for the ADC to function. [Figure 26]1 shows chemical structures of chemical entities containing the toxic payload MMAE that can be conjugated to an antibody to form an antibody-drug conjugate, also known as an ADC. MC represents a maleimidocaproyl (MC) moiety that facilitates binding to a cysteine ​​on the antibody. VC represents a valine-citrulline (VC) moiety, and PAB represents a para-aminobenzyl (PAB) moiety, both of which facilitate enzymatic cleavage in cancer cells by the lysosomal enzyme cathepsin B. MMAE represents a monomethylauristatin E (MMAE) moiety, which is a toxic payload that inhibits cell division by blocking the polymerization of tubulin, and is a toxic payload after cell internalization and / or cleavage of the non-toxic moiety. [Figure 27] 1 shows chemical structures of chemical entities including the toxic payload MMAF that can be conjugated to antibodies to form antibody-drug conjugates, also known as ADCs. MC represents the maleimidocaproyl (MC) moiety that facilitates binding to cysteines on the antibody. VC represents the valine-citrulline (VC) moiety and PAB represents the para-aminobenzyl (PAB) moiety, both of which facilitate enzymatic cleavage of the toxic payload in cancer cells by the lysosomal enzyme cathepsin B. MMAF represents the monomethylauristatin E (MMAF) moiety, which is the toxic payload after cell internalization and / or cleavage of the non-toxic moiety. Because the MMAF payload contains a carboxylic acid, it is difficult, if not impossible, for the payload to exit the cell membrane after cleavage from the antibody. Thus, it is after the payload is internalized that MMAF exhibits potent tubulin inhibition. [Figure 28]1 shows the chemical structure of an exatecan derivative, Dxd, assembled in a reactive configuration, called delxtecan, ready for conjugation to an antibody. MC represents a maleimidocaproyl (MC) moiety that facilitates attachment to a cysteine ​​on the antibody. GGFG represents a glycine-glycine-phenylalanine-glycine (GGFG) moiety that provides a flexible linker. Coupler represents a coupler (HN-CH2-O-CH2-CO) moiety that bridges the linker and payload via an ether-diamide coupler. Exatecan represents the Dxd moiety, which is the toxic payload after cellular internalization and / or cleavage of the non-toxic moiety. [Figure 29] Figure 1 shows chemical structures of exatecan derivatives incorporated with different chemistries to facilitate conjugation to antibodies. MC represents a maleimidocaproyl (MC) moiety that facilitates binding to a cysteine ​​on the antibody. VC represents a valine-citrulline (VC) moiety and PAB represents a para-aminobenzyl (PAB) moiety, both of which facilitate enzymatic cleavage of the toxic payload within cancer cells by the lysosomal enzyme cathepsin B. Exatecan represents the exatecan moiety that is the toxic payload after cellular internalization and / or cleavage of the non-toxic moiety. [Diagram 30] Figure 1 shows a hydrophobic interaction chromatography (HIC) chromatogram of MNC2 bound to MMAE. IgG1 antibodies such as MNC2 have up to eight cysteines with free thiols that can be conjugated to toxins or protoxins via the free thiols. Thus, up to eight toxins or payloads can be attached to each antibody. Analysis of the HIC chromatogram of MNC2 showed an average drug-antibody ratio (DAR) of 4.10. [Diagram 31] Figure 1 shows a hydrophobic interaction chromatography (HIC) chromatogram of MNC2 bound to MMAF. The average DAR of MNC2-MMAF was determined to be 3.65. [Figure 32A]Figure 32A shows a chromatogram of hydrophobic interaction chromatography (HIC) of MNC2 bound to deruxtecan or exatecan. Figure 32B shows a chromatogram of hydrophobic interaction chromatography (HIC) of MNC2 bound to deruxtecan. The DAR of MNC2-deruxtecan was determined to be 7.7. [Figure 32B] Figure 32B shows a chromatogram of hydrophobic interaction chromatography (HIC) of MNC2 bound to deruxtecan or exatecan. Figure 32B shows a chromatogram of hydrophobic interaction chromatography (HIC) of MNC2 bound to exatecan. The DAR of MNC2-exatecan was determined to be 8.2. [Diagram 33] 1 shows a hydrophobic interaction chromatography (HIC) chromatogram of MN20A10 bound to MMAE. The average DAR of MN20A10-MMAE was 2.96. [Diagram 34] Figure 1 shows a hydrophobic interaction chromatography (HIC) chromatogram of MN20A10 bound to MMAF. The average DAR of MN20A10-MMAF was 3.79. [Diagram 35] 1 shows a hydrophobic interaction chromatography (HIC) chromatogram of MN20A10 bound to deruxtecan. The average DAR of MN20A10-deruxtecan was 4.6. [Diagram 36] Figure 36 shows flow cytometry graphs measuring the ability of MNC2 to recognize cancer cells before and after conjugation with MMAE. Figure 36A shows the percentage of T47D breast cancer cells recognized by MNC2 before and after conjugation with MMAE. Figure 36B shows the percentage of T47D breast cancer cells recognized by MNC2 after conjugation to MMAE. Figure 36C shows the percentage of HCT-116, MUC1 negative cells recognized by MNC2 before and after conjugation with MMAE. Figure 36D shows the percentage of NCI-H1975 lung cancer cells recognized by MNC2 after conjugation with MMAE. [Figure 37]Flow cytometry graphs measuring the ability of MN20A10 to recognize cancer cells before and after conjugation with MMAE are shown. Figure 37A shows the percentage of T47D breast cancer cells recognized by MN20A10 before and after conjugation with MMAE. Figure 37B shows the percentage of T47D breast cancer cells recognized by MN20A10 after conjugation to MMAE. Figure 37C shows the percentage of HCT-116, MUC1 negative cancer cells recognized by MN20A10 before conjugation with MMAE. Figure 37D shows the percentage of NCI-H1975 lung cancer cells recognized by MN20A10 after conjugation with MMAE. [Figure 38-1] FIG. 38 shows a graph of a plate reader assay, in which the viability of target cancer cells is measured as a function of the concentration of added MNC2-ADC, with viability measured using PrestoBlue. The experiment was continued for 72 hours, after which measurements were taken. FIG. 38A shows the viability of T47D wild-type breast cancer cells after the addition of either MNC2-MMAE or MNC2-MMAF. FIG. 38B shows the viability of T47D breast cancer cells engineered to express more MUC1*, designated T47D-MUC1*, after the addition of either MNC2-MMAE or MNC2-MMAF. FIG. 38C shows the viability of HPAF II wild-type pancreatic cancer cells after the addition of either MNC2-MMAE or MNC2-MMAF. FIG. 38D shows the viability of HPAF II pancreatic cancer cells engineered to express more MUC1*, designated HPAF II-MUC1*, after the addition of either MNC2-MMAE or MNC2-MMAF. [Figure 38-2]FIG. 38 shows a graph of a plate reader assay, in which the viability of target cancer cells is measured as a function of the concentration of MNC2-ADC added, with viability measured using PrestoBlue. The experiment was allowed to continue for 72 hours before measurements were taken. FIG. 38E shows a table of calculated IC50s for the killing ability of MNC2-MMAE or MNC2-MMAF added to either breast cancer cells or pancreatic cells expressing low levels of the target antigen, MUC1*. FIG. 38F shows a table of calculated IC50s for the killing ability of MNC2-MMAE or MNC2-MMAF added to either breast cancer cells or pancreatic cells expressing high levels of the target antigen, MUC1*. [Figure 39-1] FIG. 39 shows a graph of a plate reader assay, in which the viability of target cancer cells is measured as a function of the concentration of MN20A10-ADC added, with viability measured using PrestoBlue. The experiment was allowed to continue for 72 hours before measurements were taken. FIG. 39A shows the viability of T47D wild-type breast cancer cells after addition of either MN20A10-MMAE or MN20A10-MMAF. FIG. 39B shows the viability of T47D breast cancer cells engineered to express more MUC1*, designated T47D-MUC1*, after addition of either MN20A10-MMAE or MN20A10-MMAF. FIG. 39C shows the viability of HPAF II wild-type pancreatic cancer cells after addition of either MN20A10-MMAE or MN20A10-MMAF. FIG. 39D shows the viability of HPAF II pancreatic cancer cells engineered to express more MUC1*, designated HPAF II-MUC1*, following the addition of either MN20A10-MMAE or MN20A10-MMAF. [Figure 39-2]FIG. 39A shows a graph of a plate reader assay in which target cancer cell viability is measured as a function of the concentration of MN20A10-ADC added, with viability measured using PrestoBlue. The experiment was allowed to continue for 72 hours before measurements were taken. FIG. 39E shows a table of calculated IC50s for the killing ability of MN20A10-MMAE or MN20A10-MMAF added to either breast cancer cells or pancreatic cells expressing low to moderate levels of the target antigen, MUC1*. FIG. 39F shows a table of calculated IC50s for the killing ability of MN20A10-MMAE or MN20A10-MMAF added to either breast cancer cells or pancreatic cells expressing high levels of the target antigen, MUC1*. [Diagram 40] 4 shows a graph of a plate reader assay, in which the viability of target cancer cells is measured as a function of the concentration of added MNC2-ADC or MN20A10-ADC, and viability is measured using PrestoBlue. The experiment was continued for 72 hours or 120 hours, after which measurements were taken for MMAE or deruxtecan conjugates, respectively. FIG. 40A shows the viability of T47D wild-type breast cancer cells after addition of either MNC2-MMAE, MNC2-deruxtecan, MN20A10-MMAE, or MN20A10-deruxtecan. FIG. 40B shows the viability of T47D breast cancer cells engineered to express more MUC1*, designated T47D-MUC1*, after addition of either MNC2-MMAE, MNC2-deruxtecan, MN20A10-MMAE, or MN20A10-deruxtecan. FIG. 40C shows a table of IC50 calculated for the killing potency of MNC2-MMAE, MNC2-deruxtecan, MN20A10-MMAE, or MN20A10-deruxtecan added to either T47D breast cancer cells expressing moderate to low MUC1* or T47D-MUC1* breast cancer cells expressing high MUC1*. [Diagram 41]41A shows a graph of a plate reader assay, in which the viability of target cancer cells is measured using PrestoBlue as a function of the concentration of added MNC2-ADC or MN20A10-ADC. The experiment was continued for 72 hours, after which measurements were taken. FIG. 41A shows the viability of DU145 wild-type hormone refractory prostate cancer cells after addition of either MNC2-MMAE or MN20A10-MMAE. FIG. 41B shows a table of calculated IC50 for the killing ability of MNC2-MMAE and MN20A10-MMAE in these prostate cancer cells. [Diagram 42] 42A shows a graph of a plate reader assay, in which the viability of target cancer cells is measured using PrestoBlue as a function of the concentration of added MNC2-ADC or MN20A10-ADC. The experiment was allowed to continue for 72 hours before measurements were taken. FIG. 42A shows the viability of NCI-H1975 non-small cell lung cancer cells after addition of either MNC2-MMAE or MN20A10-MMAE. FIG. 42B shows a table of calculated IC50 for the killing ability of MNC2-MMAE and MN20A10-MMAE for these lung cancer cells. [Diagram 43] FIG. 43 shows a graph of a plate reader assay in which the viability of target cancer cells is measured after addition of MNC2-MMAE or MN20A10-MMAE. The experiment was continued for 72 hours before measurements were taken. FIG. 43A shows the viability of T47D wild-type breast cancer cells after addition of either MNC2-MMAE or MN20A10-MMAE. FIG. 43B shows the viability of T47D breast cancer cells engineered to express more MUC1*, designated T47D-MUC1*, after addition of either MNC2-MMAE or MN20A10-MMAE. [Diagram 44]A graph of a plate reader assay is shown comparing the viability of MUC1* expressing cancer cells, HCT-MUC1*, to MUC1* negative cancer cells, HCT-116-wt, after treatment with either MN20A10-MMAE or the isotype control antibody IgG2b-MMAE. The experiment was continued for 72 hours before flow cytometry measurements. [Diagram 45] 45A shows a graph of a plate reader assay, in which the viability of MUC1*-expressing cancer cells is measured after addition of either unconjugated MN20A10 or MN20A10-MMAE. FIG. 45A shows the killing effect of MN20A10-MMAE on NCI H1975 non-small cell lung cancer cells. FIG. 45B shows the killing effect of MN20A10-MMAE on DU145 hormone refractory prostate cancer cells. FIG. 45C shows the killing effect of MN20A10-MMAE on wild-type HPAF II pancreatic cancer cells or HPAFI-MUC1* cancer cells engineered to express more MUC1*. FIG. 45D shows a table of IC50. [Figure 46-1] 46A shows a magnified photograph of T47D-MUC1* breast cancer cells incubated with either MNC2-MMAE or MNC2-deruxtecan. FIG. 46A is a photograph taken at 4x magnification and shows the killing effect of 500nM MNC2-MMAE on breast cancer cells after 120 hours. FIG. 46B is a photograph taken at 4x magnification and shows the killing effect of 6.2nM MNC2-MMAE on breast cancer cells after 120 hours. FIG. 46C is a photograph taken at 4x magnification of untreated T47D breast cancer cells as a control. FIG. 46D shows the killing effect of 500nM MNC2-deruxtecan on breast cancer cells after 120 hours. FIG. 46E shows the killing effect of 6.2nM MNC2-deruxtecan on breast cancer cells after 120 hours. FIG. 46F shows untreated T47D breast cancer cells as a control. [Figure 46-2]46A and 46B are magnified photographs of T47D-MUC1* breast cancer cells incubated with either MNC2-MMAE or MNC2-deruxtecan. FIG. 46G is a photograph taken at 20x magnification, showing the killing effect of 6.2nM MNC2-MMAE on breast cancer cells after 120 hours. FIG. 46H is a photograph taken at 20x magnification, showing untreated breast cancer cells as a control. FIG. 46I is a photograph taken at 20x magnification, showing the killing effect of 6.2nM MNC2-deruxtecan on breast cancer cells after 120 hours. FIG. 46J is a photograph taken at 20x magnification, showing untreated breast cancer cells as a control. The killing effect can be easily recognized as a significant decrease in cell number, a change in cell morphology to a rounding up morphology, and cell lifting. In contrast, control wells showed a confluent monolayer of compact cells with a flattened, spread morphology and no floating dead cells. [Figure 47] Figure 47 shows the photographs of DU145 prostate cancer cells incubated with either MNC2-MMAE or MNC2-deruxtecan after 120 hours, taken at 4x magnification. Figure 47A shows the enlarged photograph of the killing effect of 500nM MNC2-MMAE. Figure 47B shows the enlarged photograph of untreated cells with normal morphology and confluency. Figure 47C shows the enlarged photograph of the killing effect of 500nM MNC2-deruxtecan. Figure 47D shows the enlarged photograph of untreated cells with normal morphology and confluency. [Figure 48-1]48A shows photographs taken at magnification of T47D-MUC1* breast cancer cells incubated with either MN20A10-MMAE or MN20A10-deruxtecan. FIG. 48A is a photograph taken at 4x magnification and shows the killing effect of 500 nM MN20A10-MMAE on breast cancer cells after 120 hours. FIG. 48B is a photograph taken at 4x magnification and shows the killing effect of 56 nM MN20A10-MMAE on breast cancer cells after 120 hours. FIG. 48C is a photograph taken at 4x magnification and shows untreated T47D breast cancer cells as a control. FIG. 48D shows the killing effect of 500 nM MN20A10-deruxtecan on breast cancer cells after 120 hours. FIG. 48E shows the killing effect of 56 nM MN20A10-deruxtecan on breast cancer cells after 120 hours. FIG. 48F shows untreated T47D breast cancer cells as a control. [Figure 48-2] 48A-48J are photographs taken at magnification of T47D-MUC1* breast cancer cells incubated with either MN20A10-MMAE or MN20A10-deruxtecan. FIG. 48G is a photograph taken at 20x magnification and shows the killing effect of 56nM MN20A10-MMAE on breast cancer cells after 120 hours. FIG. 48H is a photograph taken at 20x magnification and shows untreated breast cancer cells as a control. FIG. 48I is a photograph taken at 20x magnification and shows the killing effect of 56nM MN20A10-deruxtecan on breast cancer cells after 120 hours. FIG. 48J is a photograph taken at 20x magnification and shows untreated breast cancer cells as a control. [Figure 48-3]Figure 48 shows a photograph taken at magnification of T47D-MUC1* breast cancer cells incubated with either MN20A10-MMAE or MN20A10-deruxtecan. Figure 48K shows a photograph taken at 20x magnification and DU145 prostate cancer cells treated with 500nM MN20A10-MMAE, DAR5.8. Treated cells show aggregated and dead cells. Figure 48L shows a photograph taken at 20x magnification and untreated control DU145 prostate cancer cells. Figure 48M shows a photograph taken at 20x magnification and DU145 prostate cancer cells treated with 500nM MN20A10-deruxtecan, DAR4.6. Treated cells show aggregated and dead cells. Figure 48N shows a photograph taken at 20x magnification and untreated control DU145 prostate cancer cells. [Figure 49-1]Figure 49 shows traces from a real-time killing assay performed on an xCELLigence instrument. In this assay, impedance is measured in real time. Impedance (insulation) decreases as attached cancer cells die and detach from the electrode surface. T47D wild-type breast cancer cells were plated at 5,000 cells per well in a multi-electrode well plate and allowed to attach and grow for 24 hours before adding the ADC. The cancer cells were then added with either MNC2-MMAE, DAR4.1, MN20A10-MMAE, DAR5.8, MNC2-MMAF, DAR3.7, MN20A10-MMAF, DAR3.8, MNC2-deruxtecan, DAR 7.2, or MN20A10-deruxtecan, DAR4.6. Figure 49A shows MNC2-MMAE added at a final concentration of 500 nM, 167 nM, or 2 nM, with the experimental readout being taken 40 hours after ADC addition. Taxol and triton were added as positive killing controls. Figure 49B shows MN20A10-MMAE added at a final concentration of 167 nM, 56 nM, or 2 nM, with the experimental readout occurring 40 hours after ADC addition. Figure 49C shows MNC2-MMAF added at a final concentration of 500 nM, 167 nM, or 2 nM, with the experimental readout occurring 40 hours after ADC addition. Taxol and triton were added as positive killing controls. Figure 49D shows MN20A10-MMAF added at a final concentration of 500 nM, 167 nM, or 2 nM, with the experimental readout occurring 40 hours after ADC addition. [Figure 49-2]Figure 49 shows traces from a real-time killing assay performed on an xCELLigence instrument. The assay measures impedance in real time. Impedance (insulation) decreases as attached cancer cells die and detach from the electrode surface. T47D wild-type breast cancer cells were plated at 5,000 cells per well in a multi-electrode well plate and allowed to attach and grow for 24 hours before adding the ADC. The cancer cells were then added with either MNC2-MMAE, DAR4.1, MN20A10-MMAE, DAR5.8, MNC2-MMAF, DAR3.7, MN20A10-MMAF, DAR3.8, MNC2-deruxtecan, DAR 7.2, or MN20A10-deruxtecan, DAR4.6. Figure 49E shows MNC2-MMAE added at a final concentration of 500 nM, 167 nM, or 2 nM, with the experimental readout being taken 120 hours after ADC addition. As shown, essentially all tumor cells were killed at a MNC2-MMAE concentration of 167 nM. Figure 49F shows MNC2-MMAF added at a final concentration of 500 nM, 167 nM, or 2 nM, with the experimental readout being performed 120 hours after ADC addition. As shown, essentially all tumor cells were killed at a MNC2-MMAF concentration of 167 nM. Figure 49G shows MNC2-deruxtecan added at a final concentration of 500 nM, 167 nM, or 2 nM, with the experimental readout being performed 120 hours after ADC addition. As shown, significant tumor cell killing occurs later than MNC2-MMAE or MNC2-MMAF, but by 100 hours, approximately the same level of killing is reached at a MNC2-deruxtecan concentration of 167 nM. Figure 49H shows MN20A10-MMAE added over a range of concentrations with the experimental readout being 120 hours after ADC addition. As shown, essentially all tumor cells were killed at a MN20A10-MMAE concentration of 56 nM. Figure 49I shows MN20A10-MMAF added over a range of concentrations with the experimental readout being 120 hours after ADC addition. As shown, essentially all tumor cells were killed at a MN20A10-MMAF concentration of 167 nM.Figure 49J shows MN20A10-deruxtecan added over a range of concentrations with the experimental readout being 120 hours after ADC addition. As shown, tumor cell killing occurs later than MN20A10-MMAE or MN20A10-MMAF, but by 120 hours, significant killing is seen at a MN20A10-deruxtecan concentration of 167 nM. [Figure 50-1] Figure 1 shows traces from a real-time killing assay performed on the xCELLigence instrument. In this assay, impedance is measured in real time. As attached cancer cells die and detach from the electrode surface, the impedance (insulation) decreases. T47D-MUC1* breast cancer cells engineered to express more MUC1* were plated at 5,000 cells per well in multi-electrode well plates and allowed to attach and grow for 24 hours before adding ADC. The cancer cells were then added with either MNC2-MMAE, DAR4.1, MN20A10-MMAE, DAR5.8, MNC2-MMAF, DAR3.7, MN20A10-MMAF, DAR3.8, MNC2-deruxtecan, DAR7.2, or MN20A10-deruxtecan, DAR4.6. FIG. 50A shows MNC2-MMAE added at a final concentration of 19 nM, 6 nM, or 0.69 nM, and the experimental readout is performed 40 hours after ADC addition. As can be seen, essentially all tumor cells die at 19 nM. Taxol and Triton were added as positive killing controls. FIG. 50B shows MN20A10-MMAE added at a final concentration of 19 nM, 6 nM, or 2 nM, and the experimental readout is performed 40 hours after ADC addition. As can be seen, essentially all tumor cells die at 19 nM. FIG. 50C shows MNC2-MMAF added at a final concentration of 6 nM, 2 nM, or 0.69 nM, and the experimental readout is performed 40 hours after ADC addition. As can be seen, essentially all tumor cells die at 6 nM. Taxol and Triton were added as positive killing controls. Figure 50D shows MN20A10-MMAF added at final concentrations of 56 nM, 19 nM, or 2 nM with the experimental readout being 40 hours after ADC addition. As can be seen, essentially all tumor cells are killed at 56 nM. [Figure 50-2]Figure 50 shows traces from a real-time killing assay performed on an xCELLigence instrument. In this assay, impedance is measured in real time. As attached cancer cells die and detach from the electrode surface, impedance (insulation) decreases. T47D-MUC1* breast cancer cells engineered to express more MUC1* were plated at 5,000 cells per well in multi-electrode well plates and allowed to attach and grow for 24 hours before adding ADC. The cancer cells were then added with either MNC2-MMAE, DAR4.1, MN20A10-MMAE, DAR5.8, MNC2-MMAF, DAR3.7, MN20A10-MMAF, DAR3.8, MNC2-deruxtecan, DAR7.2, or MN20A10-deruxtecan, DAR4.6. Figure 50E shows MNC2-MMAE added over a range of concentrations, with the experimental readout being taken 120 hours after ADC addition. As shown, essentially all T47D-MUC1* tumor cells were killed at a MNC2-MMAE concentration of 6 nM. Figure 50F shows MNC2-MMAF added over a range of concentrations, with the experimental readout being 120 hours after ADC addition. As shown, essentially all T47D-MUC1* tumor cells were killed at a MNC2-MMAF concentration of 2 nM. Figure 50G shows MNC2-deruxtecan added over a range of concentrations, with the experimental readout being 120 hours after ADC addition. As shown, significant T47D-MUC1* tumor cell killing occurs later than MNC2-MMAE or MNC2-MMAF, but by 120 hours, approximately the same level of killing is reached at a MNC2-deruxtecan concentration of 6 nM. Figure 50H shows MN20A10-MMAE added over a range of concentrations, with the experimental readout being 120 hours after ADC addition. As shown, essentially all tumor cells were killed at a MN20A10-MMAE concentration of 19 nM. Figure 50I shows MN20A10-MMAF added over a range of concentrations, with the experimental readout being 120 hours after ADC addition. As shown, essentially all tumor cells were killed at a MN20A10-MMAF concentration of 19 nM.Figure 50J shows MN20A10-deruxtecan added over a range of concentrations with the experimental readout being 120 hours after ADC addition. As shown, T47D-MUC1* tumor cell killing occurs later than MN20A10-MMAE or MN20A10-MMAF, but by 120 hours essentially all tumor cells were killed at a MN20A10-deruxtecan concentration of 167 nM. [Figure 51-1] Figure 51 shows traces from a real-time killing assay performed on an xCELLigence instrument. The assay measures impedance in real time. Impedance (insulation) decreases as attached cancer cells die and detach from the electrode surface. NCI-H1975 lung cancer cells were plated at 5,000 cells / well on a multi-electrode well plate and allowed to attach and grow for 24 hours before adding ADC. The cancer cells were then added with either MNC2-MMAE, DAR4.1, MN20A10-MMAE, DAR5.8, MNC2-MMAF, DAR3.7, MN20A10-MMAF, DAR3.8, MNC2-deruxtecan, DAR7.2, or MN20A10-deruxtecan, DAR4.6. Figure 51A shows MNC2-MMAE added over a range of concentrations, with the experimental readout at 40 hours. As shown, essentially all tumor cells were killed at a MNC2-MMAF concentration of 167 nM. Taxol and Triton were added as positive killing controls. Figure 51B shows MN20A10-MMAE added over a range of concentrations, with the experimental readout at 40 hours. As shown, essentially all tumor cells were killed at a MNC2-MMAF concentration of 167 nM. Figure 51C shows MNC2-MMAF added over a range of concentrations, with the experimental readout at 40 hours. As shown, essentially all tumor cells were killed at a MNC2-MMAF concentration of 167 nM. Taxol and Triton were added as positive killing controls. Figure 51D shows MN20A10-MMAF added over a range of concentrations, with the experimental readout at 40 hours. As shown, essentially all tumor cells were killed at a MNC2-MMAF concentration of 167 nM. [Figure 51-2] Figure 51 shows traces from a real-time killing assay performed on an xCELLigence instrument. The assay measures impedance in real time. Impedance (insulation) decreases as attached cancer cells die and detach from the electrode surface. NCI-H1975 lung cancer cells were plated at 5,000 cells / well on a multi-electrode well plate and allowed to attach and grow for 24 hours before adding ADC. The cancer cells were then added with either MNC2-MMAE, DAR4.1, MN20A10-MMAE, DAR5.8, MNC2-MMAF, DAR3.7, MN20A10-MMAF, DAR3.8, MNC2-deruxtecan, DAR7.2, or MN20A10-deruxtecan, DAR4.6. Figure 51E shows MNC2-MMAE added over a range of concentrations, with the experimental readout being taken 120 hours after ADC addition. As shown, essentially all lung tumor cells were killed at a MNC2-MMAE concentration of 167 nM. FIG. 51F shows MNC2-MMAF added over a range of concentrations with the experimental readout being 120 hours after ADC addition. As shown, essentially all tumor cells were killed at a MNC2-MMAF concentration of 56 nM. FIG. 51G shows MNC2-deruxtecan added over a range of concentrations with the experimental readout being 120 hours after ADC addition. FIG. 51H shows MN20A10-MMAE added over a range of concentrations with the experimental readout being 120 hours after ADC addition. As shown, essentially all tumor cells were killed at MN20A10-MMAE concentrations of 56 nM to 19 nM. FIG. 51I shows MN20A10-MMAF added over a range of concentrations with the experimental readout being 120 hours after ADC addition. As shown, essentially all tumor cells were killed at a MN20A10-MMAF concentration of 56 nM. [Figure 52-1]Figure 52 shows traces from a real-time killing assay performed on an xCELLigence instrument. The assay measures impedance in real time. Impedance (insulation) decreases as attached cancer cells die and detach from the electrode surface. HPAF II wild-type (WT) pancreatic cancer cells were plated at 5,000 cells / well on a multi-electrode well plate and allowed to attach and grow for 24 hours before adding ADC. Either MNC2-MMAE, DAR4.1, MN20A10-MMAE, DAR5.8, MNC2-MMAF, DAR3.7, MN20A10-MMAF, DAR3.8, MNC2-deruxtecan, DAR7.2, or MN20A10-deruxtecan, DAR4.6 were then added to the cancer cells. Figure 52A shows MNC2-MMAE added over a range of concentrations, with the experimental readout at 40 hours. Taxol and Triton were added as positive killing controls. Figure 52B shows MN20A10-MMAE added over a range of concentrations with the experimental readout at 40 hours. Figure 52C shows MNC2-MMAF added over a range of concentrations with the experimental readout at 40 hours. Taxol and Triton were added as positive killing controls. Figure 52D shows MN20A10-MMAF added over a range of concentrations with the experimental readout at 40 hours. [Figure 52-2]Figure 52 shows traces from a real-time killing assay performed on an xCELLigence instrument. The assay measures impedance in real time. Impedance (insulation) decreases as attached cancer cells die and detach from the electrode surface. HPAF II wild-type (WT) pancreatic cancer cells were plated at 5,000 cells / well on a multi-electrode well plate and allowed to attach and grow for 24 hours before adding ADC. The cancer cells were then added with either MNC2-MMAE, DAR4.1, MN20A10-MMAE, DAR5.8, MNC2-MMAF, DAR3.7, MN20A10-MMAF, DAR3.8, MNC2-deruxtecan, DAR7.2, or MN20A10-deruxtecan, DAR4.6. Figure 52E shows MNC2-MMAE added over a range of concentrations, with the experimental readout being taken 120 hours after ADC addition. As shown, essentially all pancreatic tumor cells were killed at a MNC2-MMAE concentration of 56 nM. FIG. 52F shows MNC2-MMAF added over a range of concentrations, with the experimental readout at 120 hours after ADC addition. As shown, essentially all tumor cells were killed at a MNC2-MMAF concentration of 56 nM. FIG. 52G shows MNC2-deruxtecan added over a range of concentrations, with the experimental readout at 120 hours after ADC addition. Significant killing is measured at 120 hours, at a concentration of about 56 nM. FIG. 52H shows MN20A10-MMAE added over a range of concentrations, with the experimental readout at 120 hours after ADC addition. As shown, essentially all tumor cells were killed at MN20A10-MMAE concentrations of 56 nM to 19 nM. Figure 52I shows MN20A10-MMAF added over a range of concentrations with the experimental readout being 120 hours after ADC addition. As shown, essentially all tumor cells were killed at a MN20A10-MMAF concentration of 56 nM. [Figure 53-1]Figure 53 shows traces from a real-time killing assay performed on an xCELLigence instrument. In this assay, impedance is measured in real time. As attached cancer cells die and detach from the electrode surface, impedance (insulation) decreases. HPAF II-MUC1* pancreatic cancer cells engineered to express more MUC1* were plated at 5,000 cells / well on a multi-electrode well plate and allowed to attach and grow for 24 hours before adding ADC. The cancer cells were then added with either MNC2-MMAE, DAR4.1, MN20A10-MMAE, DAR5.8, MNC2-MMAF, DAR3.7, MN20A10-MMAF, DAR3.8, MNC2-deruxtecan, DAR7.2, or MN20A10-deruxtecan, DAR4.6. Figure 53A shows MNC2-MMAE added over a range of concentrations, with the experimental readout at 40 hours. Taxol and triton were added as positive killing controls. Figure 53B shows MN20A10-MMAE added over a range of concentrations with the experimental readout at 40 hours. Figure 53C shows MNC2-MMAF added over a range of concentrations with the experimental readout at 40 hours. Taxol and triton were added as positive killing controls. Figure 53D shows MN20A10-MMAF added over a range of concentrations with the experimental readout at 40 hours. [Figure 53-2]Figure 1 shows traces from a real-time killing assay performed on the xCELLigence instrument. In this assay, impedance is measured in real time. Impedance (insulation) decreases as attached cancer cells die and detach from the electrode surface. HPAF II-MUC1* pancreatic cancer cells engineered to express more MUC1* were plated at 5,000 cells / well on multi-electrode well plates, allowed to attach, and allowed to attach and grow for 24 hours before adding ADC. Either MNC2-MMAE, DAR4.1, MN20A10-MMAE, DAR5.8, MNC2-MMAF, DAR3.7, MN20A10-MMAF, DAR3.8, MNC2-deruxtecan, DAR7.2, or MN20A10-deruxtecan, DAR4.6 were then added to the cancer cells. FIG. 53E shows MNC2-MMAE added over a range of concentrations with the experimental readout being 120 hours after ADC addition. As shown, essentially all of the pancreatic tumor cells were killed at a MNC2-MMAE concentration of 56 nM. FIG. 53F shows MNC2-MMAF added over a range of concentrations with the experimental readout being 120 hours after ADC addition. As shown, essentially all of the tumor cells were killed at a MNC2-MMAF concentration of 56 nM. FIG. 53G shows MNC2-deruxtecan added over a range of concentrations with the experimental readout being 120 hours after ADC addition. As can be seen, by 120 hours, nearly all of the pancreatic cancer cells are killed at a concentration of 167 nM. FIG. 53H shows MN20A10-MMAE added over a range of concentrations with the experimental readout being 120 hours after ADC addition. As shown, essentially all tumor cells were killed at MN20A10-MMAE concentrations between 56 nM and 19 nM. Figure 53I shows MN20A10-MMAF added over a range of concentrations, with the experimental readout being 120 hours after ADC addition. As shown, essentially all tumor cells were killed at a MN20A10-MMAF concentration of 56 nM. [Figure 54]Figure 54 shows traces from a real-time killing assay performed on the xCELLigence instrument. In this assay, impedance is measured in real time. Impedance (insulation) decreases as attached cancer cells die and detach from the electrode surface. Here, T47D-wt breast cancer cells expressing low to moderate levels of MUC1* were plated at 5,000 cells per well on a multi-electrode 96-well plate and allowed to attach and grow for 24 hours before adding the ADC. The cancer cells were then added with either MNC2-exatecan, DAR8.2, MNC2-deruxtecan, DAR4.0, MNC2-MMAE, DAR4.1, or 50 μM free exatecan. xCELLigence readout is from 0 hours to 120 hours. Figure 54A shows various ADCs added at a concentration of 500 nM. Figure 54B shows various ADCs added at a concentration of 167 nM. Figure 54C shows various ADCs added at a concentration of 56 nM. Figure 54D shows various ADCs added at a concentration of 19 nM. Figure 54E shows a key that lists the colors of the traces for each ADC and their respective DARs. As can be seen, MNC2-exatecan potently kills target cancer cells at concentrations as low as 56 nM. [Figure 55]Figure 55 shows traces from a real-time killing assay performed on the xCELLigence instrument. In this assay, impedance is measured in real time. As attached cancer cells die and detach from the electrode surface, impedance (insulation) drops. Here, T47D-MUC1* breast cancer cells engineered to express high levels of MUC1* were plated at 5,000 cells per well on a multi-electrode 96-well plate and allowed to adhere and grow for 24 hours before adding the ADC. The cancer cells were then added with either MNC2-exatecan, DAR8.2, MNC2-deruxtecan, DAR4.0, MNC2-MMAE, DAR4.1, or 50 μM free exatecan. xCELLigence readout is from 0 hours to 120 hours. Figure 55A shows various ADCs added at a concentration of 19 nM. Figure 55B shows various ADCs added at a concentration of 6.2 nM. Figure 55C shows various ADCs added at a concentration of 2.1 nM. Figure 55D shows various ADCs added at a concentration of 0.69 nM. Figure 55E shows a key that lists the color of the traces for each ADC and their respective DARs. As can be seen, MNC2-exatecan and MNC2-deruxtecan potently kill cancer cells expressing high levels of the target antigen at concentrations as low as 6.2 nM and even 2.1 nM. [Figure 56]Figure 56 shows traces from a real-time killing assay performed on the xCELLigence instrument. In this assay, impedance is measured in real time. As attached cancer cells die and detach from the electrode surface, impedance (insulation) drops. Here, NCI-H1975 non-small cell lung cancer cells expressing very low levels of MUC1* were plated at 5,000 cells per well on a multi-electrode 96-well plate and allowed to adhere and grow for 24 hours before adding the ADC. The cancer cells were then added with either MNC2-exatecan, DAR8.2, MNC2-deruxtecan, DAR4.0, MNC2-MMAE, DAR4.1, or 50 μM free exatecan. xCELLigence readout is from 0 hours to 120 hours. Figure 56A shows various ADCs added at a concentration of 500 nM. Figure 56B shows various ADCs added at a concentration of 167 nM. Figure 56C shows various ADCs added at a concentration of 56 nM. Figure 56D shows various ADCs added at a concentration of 19 nM. Figure 56E shows a key that lists the colors of the traces for each ADC and their respective DARs. As can be seen, MNC2-exatecan potently kills cancer cells expressing very low levels of the target antigen at concentrations as low as 167 nM. [Figure 57-1]Figure 57 shows bioluminescence photographs of female NOD / SCID / GAMMA (NSG) mice implanted with a 90 day estrogen pellet and then implanted in the right flank with 1M human breast cancer cells, either T47D wild type cells expressing low to moderate levels of MUC1*, or T47D-MUC1* cells engineered to express more MUC1*. Seven days after tumor implantation, animals were injected with MNC2-MMAE, DAR3.9 at 5mg / kg, but increased to 10mg / kg for injections on days 14 and 20. Figure 57A shows a control animal implanted with T47D-wt tumor cells but mock injected with PBS. Figure 57B shows an animal implanted with T47D-wt cells and then injected with MNC2-MMAE. Figure 57C shows a control animal implanted with T47D-MUC1* tumor cells but mock injected with PBS. FIG. 57D shows animals transplanted with T47D-MUC1* cells and then injected with MNC2-MMAE. [Figure 57-2] Figure 57B shows bioluminescence photographs of female NOD / SCID / GAMMA (NSG) mice implanted with 90-day estrogen pellets and then implanted in the right flank with 1M human breast cancer cells, either T47D wild-type cells expressing low-to-moderate levels of MUC1*, or T47D-MUC1* cells engineered to express more MUC1*. On day 7 after tumor implantation, animals were injected with MNC2-MMAE, DAR3.9 at 5 mg / kg, increasing to 10 mg / kg for injections on days 14 and 20. Figure 57E shows a graph of IVIS measurements of bioluminescence (radiance photons / cm2) as a function of days after tumor implantation for mice implanted with T47D wild-type breast cancer cells. Figure 57F shows a graph of IVIS measurements of bioluminescence (radiance photons / cm2) as a function of days after tumor implantation for mice implanted with T47D-MUC1* breast cancer cells. [Figure 58-1]Figure 58 shows bioluminescence photographs of female NOD / SCID / GAMMA (NSG) mice implanted with a 90 day estrogen pellet and then implanted in the right flank with 1M human breast cancer cells, either T47D wild type cells expressing low to moderate levels of MUC1*, or T47D-MUC1* cells engineered to express more MUC1*. Seven days after tumor implantation, animals were injected with MN20A10-MMAE, DAR3.0 at 5mg / kg, but increased to 10mg / kg for injections on days 14 and 20. Figure 58A shows a control animal implanted with T47D-wt tumor cells but mock injected with PBS. Figure 58B shows an animal implanted with T47D-wt cells and then injected with MN20A10-MMAE. Figure 58C shows a control animal implanted with T47D-MUC1* tumor cells but mock injected with PBS. FIG. 58D shows animals implanted with T47D-MUC1* cells and then injected with MN20A10-MMAE. [Figure 58-2] Figure 58B shows bioluminescence photographs of female NOD / SCID / GAMMA (NSG) mice implanted with 90-day estrogen pellets and then implanted in the right flank with 1M human breast cancer cells, either T47D wild-type cells expressing low-to-moderate levels of MUC1*, or T47D-MUC1* cells engineered to express more MUC1*. On day 7 after tumor implantation, animals were injected with MN20A10-MMAE, DAR3.0 at 5 mg / kg, increasing to 10 mg / kg for injections on days 14 and 20. Figure 58E shows a graph of IVIS measurements of bioluminescence (radiance photons / cm2) as a function of days after tumor implantation for mice implanted with T47D wild-type breast cancer cells. Figure 58F shows a graph of IVIS measurements of bioluminescence (radiance photons / sec / cm2) as a function of days after tumor implantation for mice implanted with T47D-MUC1* breast cancer cells. [Figure 59]Bioluminescence photographs of female nu / nu mice implanted with 1M human NCI-H1975 non-small cell lung cancer cells expressing low to moderate levels of MUC1* in the right flank. Seven days after tumor implantation, animals were injected with either 5 mg / kg or 10 mg / kg MNC2-MMAE as indicated. Increasing the dose of MNC2-MMAE, DAR3.9, to 10 mg / kg on days 19 and 27 increased tumor cell killing as seen in the bioluminescence photographs. Figure 59A shows a control animal implanted with NCI-H1975 non-small cell lung cancer cells but mock injected with PBS. Figure 59B shows an animal implanted with NCI-H1975 non-small cell lung cancer cells and then injected with MNC2-MMAE. Figure 59C shows a graph of IVIS measurements of bioluminescence (radiance photons / sec / cm2) as a function of days after tumor implantation. [Figure 60] Figure 60 shows bioluminescence photographs taken with an IVIS instrument of female nu / nu mice implanted with 1M human NCI-H1975 non-small cell lung cancer cells expressing low to moderate levels of MUC1* in the right flank. On days 7 and 14 after tumor implantation, animals were injected with 5mg / kg MN20A10-MMAE, DAR3.0. On day 19, the dose was increased to 10mg / kg and administered by intraperitoneal (ip) injection. As can be seen in the photographs and graphs, increasing the dose dramatically increased tumor cell killing. Figure 60A shows a control animal implanted with NCI-H1975 non-small cell lung cancer cells but mock injected with PBS. Figure 60B shows an animal implanted with NCI-H1975 non-small cell lung cancer cells and then injected with MN20A10-MMAE. Figure 60C shows a photograph and weight of tumors excised from a control animal on day 26. FIG. 60D shows a graph of IVIS measurements of bioluminescence (radiance photons / sec / cm2) as a function of days after tumor implantation. [Figure 61-1]Figure 61 shows bioluminescence photographs taken with an IVIS instrument of female nu / nu mice implanted with 0.5M human pancreatic cancer in the right flank, either HPAF II wild type (wt) cells expressing low to moderate levels of MUC1*, or HPAF II-MUC1* cells engineered to express more MUC1*. Seven days after tumor implantation, animals were injected with either PBS as a control, or 10 mg / kg MNC2-MMAE, DAR4.1. Figure 61A shows an IVIS bioluminescence photograph of a control animal implanted with HPAF II-wt pancreatic cancer cells but mock-injected with PBS. Figure 61B shows an IVIS bioluminescence photograph of an animal implanted with HPAF II-wt pancreatic cancer cells and then injected with MNC2-MMAE. Figure 61C shows an IVIS bioluminescence photograph of a control animal implanted with HPAF II-MUC1* pancreatic cancer cells but mock-injected with PBS. FIG. 61D shows IVIS bioluminescence pictures of animals implanted with HPAF II-MUC1* pancreatic cancer cells and then injected with MNC2-MMAE. [Figure 61-2] Figure 61 shows bioluminescence photographs taken with an IVIS instrument of female nu / nu mice implanted with 0.5M human pancreatic cancer in the right flank, either HPAF II wild type (wt) cells expressing low to moderate levels of MUC1*, or HPAF II-MUC1* cells engineered to express more MUC1*. Seven days after tumor implantation, animals were injected with either PBS as a control, or 10 mg / kg MNC2-MMAE, DAR4.1. Figure 61E shows a photograph of a tumor excised from an HPAF II-wt control animal that had to be sacrificed due to excessive tumor volume. Figure 61F shows a photograph of a tumor excised from an HPAF II-MUC1* control animal that had to be sacrificed due to excessive tumor volume. Figure 61G shows an overlay graph of IVIS measurements of bioluminescence (radiance photons / sec / cm2) as a function of days after tumor implantation for both control and MNC2-MMAE treated mice. IVIS data points for day 25 are omitted due to instrument failure. Figure 61H shows a bar graph of caliper measurements of tumors on day 25. Caliper measurements eliminate differences in luciferase expression levels in HPAF II-wt versus HPAF II-MUC1* cell lines. [Figure 61-3] Bioluminescence photographs taken with an IVIS instrument are shown of female nu / nu mice implanted with 0.5M human pancreatic cancer in the right flank, either HPAF II wild type (wt) cells expressing low to moderate levels of MUC1*, or HPAF II-MUC1* cells engineered to express more MUC1*. Seven days after tumor implantation, animals were injected with either PBS as a control, or 10 mg / kg MNC2-MMAE, DAR4.1. Figure 61I shows a photograph of a control animal implanted with HPAF II-wt pancreatic cancer cells but mock-injected with PBS at day 11. Figure 61J shows a photograph of a control animal implanted with HPAF II-wt pancreatic cancer cells but mock-injected with PBS at day 18. Figure 61K shows a photograph of a control animal implanted with HPAF II-wt pancreatic cancer cells but mock-injected with PBS at day 25, showing an increasingly larger tumor in the right flank. Figure 61L shows a photograph of animals implanted with HPAF II-wt pancreatic cancer cells and then injected with MNC2-MMAE on day 11. Figure 61M shows a photograph of animals implanted with HPAF II-wt pancreatic cancer cells and then injected with MNC2-MMAE on day 18. Figure 61N shows a photograph of animals implanted with HPAF II-wt pancreatic cancer cells and then injected with MNC2-MMAE on day 25, with three of the mice showing small, barely visible tumors. [Figure 61-4]Bioluminescence photographs taken with an IVIS instrument are shown of female nu / nu mice implanted with 0.5M human pancreatic cancer in the right flank, either HPAF II wild type (wt) cells expressing low to moderate levels of MUC1*, or HPAF II-MUC1* cells engineered to express more MUC1*. Seven days after tumor implantation, animals were injected with either PBS as a control, or 10 mg / kg MNC2-MMAE, DAR4.1. Figure 61O shows a photograph of a control animal implanted with HPAF II-MUC1* pancreatic cancer cells but mock injected with PBS at day 11. Figure 61P shows a photograph of a control animal implanted with HPAF II-MUC1* pancreatic cancer cells but mock injected with PBS at day 18. Figure 61Q shows a photograph of a control animal implanted with HPAF II-MUC1* pancreatic cancer cells but mock injected with PBS at day 25, showing a large tumor in the right flank. Figure 61R shows a photograph of an animal implanted with HPAF II-MUC1* pancreatic cancer cells and then injected with MNC2-MMAE on day 11. Figure 61S shows a photograph of an animal implanted with HPAF II-MUC1* pancreatic cancer cells and then injected with MNC2-MMAE on day 18. Figure 61T shows a photograph of an animal implanted with HPAF II-MUC1* pancreatic cancer cells and then injected with MNC2-MMAE on day 25, where no tumor is visible or palpable. [Figure 62]Figure 62A shows bioluminescence photographs taken with an IVIS device of female nu / nu mice implanted with 0.5M human pancreatic cancer cells, HPAF II-MUC1* cells, engineered to express more MUC1*, in the right flank. Seven days after tumor implantation, the animals were injected with either PBS as a control, or 10 mg / kg MNC2-MMAE, DAR4.1. As can be seen, by day 66, the treated mice had no tumors or only small residual tumors. The HPAF II cell line does not express luciferase well, so bioluminescence is weak. Therefore, we also performed caliper measurements to demonstrate the actual size of the tumor. Figure 62A shows IVIS bioluminescence photographs of control animals implanted with HPAF II-MUC1* pancreatic cancer cells, but mock-injected with PBS. Figure 61B shows IVIS bioluminescence photographs of animals implanted with HPAF II-MUC1* pancreatic cancer cells and then injected with 10 mg / kg MNC2-MMAE. Figure 62C shows a photograph of excised tumors from HPAF II-MUC1* control animals that had to be sacrificed due to excessive tumor volume, whereas little or no tumor remained in the MNC2-MMAE treated group. Figure 62D shows an overlay graph of IVIS measurements of bioluminescence (radiance photons / sec / cm2) as a function of days after tumor implantation for both control and MNC2-MMAE treated mice. Figure 62E shows a Kaplan-Meier survival plot of control vs. treated mice. Figure 62F shows a bar graph of caliper measurements of control vs. treated animals. [Figure 63]Bioluminescence photographs of female NOD / SCID / GAMMA (NSG) mice are shown, implanted with a 90-day estrogen pellet and then implanted with 1M human breast cancer cells in the right flank, either T47D wild type cells expressing low to moderate levels of MUC1*, or T47D-MUC1* cells engineered to express more MUC1*. Six days after tumor implantation, animals were injected with 10 mg / kg MNC2-deruxtecan, DAR4.2, but increased to 20 mg / kg for T47D-wt treated mice. Treatment for T47D-MUC1* treated mice remained constant at 10 mg / kg. Figure 63A shows a control animal implanted with T47D-wt tumor cells, but mock injected with PBS. Figure 63B shows an animal implanted with T47D-wt cells and then injected with MNC2-deruxtecan. Figure 63C shows control animals implanted with T47D-MUC1* tumor cells but mock-injected with PBS. Figure 63D shows animals implanted with T47D-MUC1* cells and then injected with MNC2-deruxtecan. Figure 63E shows a graph of IVIS measurements of bioluminescence (radiance photons / cm2) as a function of days after tumor implantation for mice implanted with T47D wild-type breast cancer cells. Figure 63F shows a bar graph of bioluminescence measurements of each mouse by day for animals implanted with T47D-wt breast cancer cells. Figure 63G shows a graph of IVIS measurements of bioluminescence (radiance photons / sec / cm2) as a function of days after tumor implantation for mice implanted with T47D-MUC1* breast cancer cells. Figure 63H shows a bar graph of bioluminescence measurements of each mouse by day for animals implanted with T47D-MUC1* breast cancer cells. [Figure 64-1]Line graphs of IVIS luminescence measurements of individual mice from day 4 to day 30 are shown. Either T47D-wt breast cancer cells or T47D-MUC1* cells expressing more MUC1* were implanted into NOD / SCOD / GAMMA mice. Mice in both groups were mock treated with PBS or treated with MNC2-deruxtecan. Six days after tumor implantation, animals were injected with 10 mg / kg MNC2-deruxtecan, DAR4.2, which was increased to 20 mg / kg for T47D-wt treated mice. Treatment for T47D-MUC1* treated mice remained constant at 10 mg / kg. Figure 64A shows the growth of T47D-wt tumors in mouse number 1 of the control group. Figure 64B shows the growth of T47D-wt tumors in mouse number 2 of the control group. Figure 64C shows the growth of T47D-wt tumor in mouse number 3 of the control group. Figure 64D shows the growth of T47D-wt tumor in mouse number 4 of the control group. Figure 64E shows the growth of T47D-wt tumor in mouse number 5 of the control group. Figure 64F shows the growth of T47D-wt tumor in mouse number 1 of the group treated with MNC2-deruxtecan. Figure 64G shows the growth of T47D-wt tumor in mouse number 2 of the group treated with MNC2-deruxtecan. Figure 64H shows the growth of T47D-wt tumor in mouse number 3 of the group treated with MNC2-deruxtecan. Figure 64I shows the growth of T47D-wt tumor in mouse number 4 of the group treated with MNC2-deruxtecan. FIG. 64J shows the growth of T47D-wt tumor in mouse number 5 in the group treated with MNC2-deruxtecan. [Figure 64-2]Line graphs of IVIS luminescence measurements of individual mice from day 4 to day 30 are shown. Either T47D-wt breast cancer cells or T47D-MUC1* cells expressing more MUC1* were implanted into NOD / SCOD / GAMMA mice. Mice in both groups were mock treated with PBS or treated with MNC2-deruxtecan. Six days after tumor implantation, animals were injected with 10 mg / kg MNC2-deruxtecan, DAR4.2, but increased to 20 mg / kg for T47D-wt treated mice. Treatment for T47D-MUC1* treated mice remained constant at 10 mg / kg. Figure 64K shows the growth of T47D-MUC1* tumors in mouse number 1 of the control group. Figure 64L shows the growth of T47D-MUC1* tumors in mouse number 2 of the control group. FIG. 64M shows the growth of T47D-MUC1* tumor in mouse number 3 of the control group. FIG. 64N shows the growth of T47D-MUC1* tumor in mouse number 4 of the control group. FIG. 64O shows the growth of T47D-MUC1* tumor in mouse number 5 of the control group. FIG. 64P shows the growth of T47D-MUC1* tumor in mouse number 1 of the group treated with MNC2-deruxtecan. FIG. 64Q shows the growth of T47D-MUC1* tumor in mouse number 2 of the group treated with MNC2-deruxtecan. FIG. 64R shows the growth of T47D-MUC1* tumor in mouse number 3 of the group treated with MNC2-deruxtecan. FIG. 64S shows the growth of T47D-MUC1* tumor in mouse number 4 of the group treated with MNC2-deruxtecan. FIG. 64T shows the growth of T47D-MUC1* tumors in mouse number 5 in the MNC2-deruxtecan treated group. [Figure 65]Images of tumor cells are shown. Staining of day 0 cells shows that the cells express more full-length MUC1 than MUC1*, with weaker staining intensity and fewer MUC1* receptors, indicative of early stage cancer. It can be easily recognized that 62 days after tumor implantation, which corresponds to about 7 years in human time, MUC1* expression has changed dramatically in late stage tumors, in terms of the extent of expression and the intensity of expression, from low to high. In contrast, staining of serial sections of tumors shows that full-length MUC1 is expressed, which would be expected since it is cleaved to MUC1* after surface expression. However, the intensity of staining has not increased, indicating that the majority of expressed MUC1 has been cleaved to the growth factor receptor form MUC1* in late stage tumors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] This application relates to anti-MUC1* multispecific antibodies and anti-MUC1* antibody conjugates and methods of making and using them. A truncated form of the MUC1 transmembrane protein is a growth factor receptor that drives the proliferation of more than 75% of all human cancers. The truncated form of MUC1, MUC1* (pronounced mu-ku-1-star), is a growth factor receptor. By cleaving and releasing a large portion of the extracellular domain of MUC1, it exposes a binding site for activation of the ligand dimers NME1, NME6, NME7, NME7AB, NME7-X1, or NME8. It is aberrantly expressed in over 75% of all cancers and is likely overexpressed in a particularly high percentage of metastatic cancers, making it an ideal target for cancer drugs (Mahanta et al. (2008) A Minimal Fragment of MUC1 Mediates Growth of Cancer Cells. PLoS ONE 3(4):e2054. doi:10.1371 / journal.pone.0002054; Fessler et al. (2009), “MUC1* is a determinant of trastuzumab (Herceptin) resistance in breast cancer cells,” Breast Cancer Res Treat. 118(1):113-124). After MUC1 cleavage, most of its extracellular domain is shed from the surface of the cell. The remaining part, MUC1*, has a truncated extracellular domain that contains most or all of the primary growth factor receptor sequence, termed PSMGFR (SEQ ID NO: 133).

[0038] Many drugs currently administered parenterally to patients are not targeted, resulting in systemic delivery of the drug to cells and tissues of the body where delivery is unnecessary and often undesirable, which can result in adverse side effects and often limits the dose of drug that can be administered (e.g., chemotherapeutic (anti-cancer), cytotoxic, enzyme inhibitor, antiviral, antibacterial).

[0039] A primary objective is to develop methods for specifically targeting therapeutic agents to cells and tissues. The benefits of such treatment include avoiding the general physiological effects of inappropriate delivery of such agents to other cells and tissues. The present disclosure overcomes these and other limitations and problems of the prior art.

[0040] Certain terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the claimed subject matter belongs. All patents, patent applications, published applications and publications, GENBANK sequences, websites, and other published materials mentioned throughout this disclosure are incorporated by reference in their entirety unless otherwise stated. Generally, the procedures for cell culture, cell infection, antibody production, and molecular biology methods are methods commonly used in the art. Such standard techniques can be found in reference books such as, for example, Sambrook et al. (2000) and Ausubel et al. (1994).

[0041] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and / or" unless specifically stated otherwise. Additionally, the use of the term "including," as well as other forms (e.g., "include," "includes," and "included") are not limiting.

[0042] The transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The transitional phrase "consisting of" excludes any element, step, or ingredient not specified. The transitional phrase "consisting essentially of" limits the scope of a claim to certain materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0043] As used herein, ranges and amounts can be expressed as about a particular value or range. About includes the exact amount. For example, "about 1 mg" means "about 1 mg" and also means "1 mg." The terms "about" and "approximately" include amounts that are generally expected to be within experimental error.

[0044] The terms "individual," "patient," or "subject" are used interchangeably. As used herein, they refer to any mammal (i.e., a species of any order, family, and genus within the taxonomic classification, kingdom Animalia, phylum Chordata, phylum Vertebrata, Mammalia). In some embodiments, the mammal is a human. None of these terms require or are limited to situations characterized by the supervision (e.g., constant or intermittent) of a medical professional (e.g., a physician, registered nurse, nurse practitioner, physician's assistant, janitorial staff, or hospice personnel).

[0045] The terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term applies to naturally occurring amino acid polymers and to amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs). The term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked by covalent peptide bonds.

[0046] Where an amino acid sequence is provided herein, L-, D-, or β-amino acid versions of the sequence are contemplated, as well as retro, inversion, and retro-inversion isoforms. Peptides also include amino acid polymers in which one or more amino acid residues are artificial chemical analogs of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. In addition, the term applies to amino acids linked by peptide bonds or other modified linkages, e.g., where the peptide bond is replaced by an α-ester, β-ester, thioamide, phosphonamide, carbamate, hydroxylate, etc. (see, e.g., Spatola, (1983) Chem. Biochem. Amino Acids and Proteins 7:267-357), where the amide is replaced by a saturated amine (see, e.g., Skiles et al., U.S. Pat. No. 4,496,542, and Kaltenbronn et al., (1990) Pp. 969-970 in Proc. 11th American Peptide Symposium, ESCOM Science Publishers, The Netherlands, which are incorporated herein by reference).

[0047] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acids are classified as hydrophobic, polar, nonpolar, and charged amino acids. Hydrophobic amino acids include small hydrophobic amino acids and large hydrophobic amino acids. Small hydrophobic amino acids can be glycine, alanine, proline, and analogs thereof. Large hydrophobic amino acids can be valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and analogs thereof. Polar amino acids can be serine, threonine, asparagine, glutamine, cysteine, tyrosine, and analogs thereof. Nonpolar amino acids can be glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, and analogs thereof. Charged amino acids can be lysine, arginine, histidine, aspartate, glutamate, and their analogs. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbon attached to hydrogen, carboxyl group, amino group, and R group, e.g., homoserine, norleucine, methionine sulfoxide. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of amino acids, but function similarly to naturally occurring amino acids. Amino acids can be either D-amino acids or L-amino acids.

[0048] As used in this specification and the appended claims, unless specified to the contrary, the following terms have the meanings indicated below.

[0049] In some embodiments, the compounds disclosed herein contain one or more asymmetric centers, thus giving rise to enantiomers, diastereomers, and other stereoisomeric forms defined in terms of absolute stereochemistry as (R) or (S). Unless otherwise specified, all stereoisomeric forms of the compounds disclosed herein are intended to be contemplated by the present disclosure. In the case where the compounds described herein contain an alkene double bond, and unless otherwise specified, the present disclosure is intended to include both E and Z geometric isomers (e.g., cis or trans). Thus, the compounds provided herein may be enantiomerically pure or may be a mixture of stereoisomers or diastereoisomers. The compounds provided herein may contain chiral centers. Such chiral centers may be in either the (R) or (S) configuration, or may be a mixture thereof. The chiral centers of the compounds provided herein may undergo epimerization in vivo. Thus, one of ordinary skill in the art will recognize that administration of a compound in its (R) form is equivalent to administration of the compound in its (S) form for compounds that undergo epimerization in vivo. Similarly, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms, are intended to be included. The term "geometric isomer" refers to the E or Z geometric isomers (e.g., cis or trans) of the alkene double bond. The term "positional isomer" refers to structural isomers around a central ring, such as ortho, meta, and para isomers around a benzene ring.

[0050] "Tautomer" refers to a molecule capable of proton transfer from one atom of the molecule to another atom of the same molecule. The compounds presented herein, in certain embodiments, exist as tautomers. In situations where tautomerization is possible, a chemical equilibrium of tautomers exists. The exact ratio of tautomers depends on a variety of factors, including physical conditions, temperature, solvent, and pH. Some examples of tautomeric equilibrium include:

[0051] [ka] Examples include:

[0052] "Pharmaceutically acceptable salt" includes both acid and base addition salts. The pharmaceutically acceptable salt of any one of the compounds or conjugates described herein is intended to include any pharmaceutically suitable salt form. The preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0053] As used herein, the terms "antibody" and "immunoglobulin" are terms of the art and may be used interchangeably herein to refer to a molecule having an antigen-binding site that specifically binds to an antigen. In some embodiments, the isolated antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, specifically binds to a protein of interest.

[0054] Examples of antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain / antibody heavy chain pairs, antibodies having two light chain / heavy chain pairs (e.g., homologous pairs), intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, bivalent antibodies (including monospecific bivalent antibodies or bispecific bivalent antibodies), single chain antibodies, or single chain variable fragments (scFv), camelized antibodies, affybodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (e.g., Anti-anti-Id antibodies), and epitope-binding fragments of any of the above.

[0055] An antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, the antibodies described herein are IgG antibodies (e.g., human IgG), or a class (e.g., human IgG1, IgG2, IgG3, or IgG4) or subclass thereof.

[0056] The CDR sequences of the antibodies disclosed herein, or the anti-MUC1* or anti-CD3 binding domain sequences disclosed herein may be defined or determined according to the following. (i) the Kabat numbering system (Kabat et al. (197) Ann. NY Acad. Sci. 190:382-391, and Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), or (ii) the Chothia numbering scheme (e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al, 1997, J. Mol. Biol., 273:927-948; Chothia et al, 1992, J. Mol. Biol., 227:799-817; Tramontano A et al, 1999, J. Mol. Biol., 227:799-817), hereinafter referred to as "Chothia CDRs". al, 1990, J. Mol. Biol. 215(1):175-82, and U.S. Patent No. 7,709,226), or (iii) the ImMunoGeneTics (IMGT) numbering system, e.g., as described in Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al, 1999, Nucleic Acids Res., 27:209-212 (“IMGT CDRs”), or (iv) MacCallum et al., 1996, J. Mol. Biol., 262:732-745. See, e.g., Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer--Verlag, Berlin (2001).

[0057] With respect to the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acid positions 31-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3), which may optionally include one or two additional amino acids following 35 (referred to as 35A and 35B in the Kabat numbering scheme). Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3). As will be appreciated by those skilled in the art, using the Kabat numbering system, the actual linear amino acid sequence of an antibody variable domain may contain fewer or additional amino acids due to shortening or elongation of FRs and / or CDRs, and therefore the Kabat number of an amino acid is not necessarily the same as its linear amino acid number.

[0058] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0059] The term "multispecific" means that an antibody is capable of specifically binding to two or more different antigenic determinants, e.g., two binding sites formed by pairs of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) that bind different antigens.

[0060] As used herein, the term "human antibody" or "humanized antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). In some cases, human antibodies are also produced in transgenic animals (e.g., mice) that can produce a full repertoire or selection of human antibodies upon immunization in the absence of endogenous immunoglobulin production. Transfer of human germline immunoglobulin gene arrays into such germline mutant mice results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al, Nature 362 (1993) 255-258; Bruggemann, M., et al, Year Immunol. 7 (1993) 33-40). In a further example, human antibodies have also been produced in phage display libraries (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al, J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole, et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al, J. Immunol. 147 (1991) 86-95).

[0061] As used herein, an "antigen" is a moiety or molecule that contains an epitope that an antibody can specifically bind to. Thus, an antigen is specifically bound by an antibody. In certain embodiments, the antigen that the antibody described herein binds to is a protein of interest, such as MUC1*, CD3, or fragments thereof.

[0062] As used herein, the term "heavy chain", when used in reference to an antibody, can refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0063] As used herein, the term "light chain" when used in reference to an antibody can refer to any of the different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0064] As used herein, the term "percent (% sequence identity)" or "sequence identity" in reference to a sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS ALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0065] In the context of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively referred to as an amino acid sequence A having or containing a particular % amino acid sequence identity with a given amino acid sequence B) is hereinafter 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of A and B in that program, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0066] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in a substantially intact form, and not an antibody fragment as defined below, and specifically refer to an antibody having a heavy chain that includes an Fc region.

[0067] An "antibody fragment" comprises only a portion of an intact antibody, which portion retains at least one, two, three, and most, or all of the functions normally associated with that portion when present in the intact antibody. In one embodiment, an antibody fragment comprises the antigen-binding site of the intact antibody and thus retains the ability to bind to antigen.

[0068] As used herein, "epitope" is a term known in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope may be a linear epitope of contiguous amino acids or may include amino acids from two or more non-contiguous regions of an antigen.

[0069] As used herein, the terms "binds," "binds to," "specifically binds," or "specifically binds to" in the context of antibody binding refer to antibody binding to an antigen (e.g., an epitope) as such binding is understood by one of skill in the art. In certain embodiments, a molecule that specifically binds to an antigen is one that is characterized by the affinity (K d ) at least 2 log, 2.5 log, 3 log, 4 log lower (higher) K d In another specific embodiment, a molecule that specifically binds to an antigen does not cross-react with other proteins.

[0070] A "linking moiety" or "linker" (e.g., denoted as L) is a molecule having two reactive ends, one for attachment to a polypeptide (e.g., an antibody) via a conjugation moiety Y, and the other for conjugation to a linking moiety (denoted as SP) or, if SP is not present, to a portion of T. The polypeptide conjugate reactive end of the linker is typically a site capable of attachment to a polypeptide (e.g., an antibody) via a cysteine ​​thiol group on the polypeptide (e.g., an antibody), and thus is typically a thiol-reactive group such as maleimide or dibromomaleimide, or a thiol-reactive group as defined herein.

[0071] As used herein, "treatment," "treating," "palliating," or "ameliorating" are used interchangeably. These terms refer to an approach to obtain a beneficial or desired result, including but not limited to therapeutic benefit and / or prophylactic benefit. "Therapeutic benefit" refers to eradication or amelioration of the underlying disease being treated. Moreover, therapeutic benefit is achieved by eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease, such that an improvement is observed in the patient, even though the patient still suffers from the underlying disease. In prophylactic benefit, the composition is administered in some embodiments to a patient who is at risk of developing a particular disease or who reports one or more of the physiological symptoms of the disease, even if the disease has not been diagnosed.

[0072] MUC1*-binding domain In some embodiments, disclosed herein are antibodies comprising an anti-MUC1* binding domain. In some embodiments, the MUC1* binding domain comprises an antibody or an antigen-binding fragment or variant thereof. In some embodiments, the antibody or an antigen-binding fragment or variant thereof is a monoclonal antibody. In some embodiments, the antibody or an antigen-binding fragment or variant thereof is a human antibody, a murine antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the MUC1* binding domain comprises a monovalent Fab, a bivalent Fab'2, a single chain variable fragment (scFv), or a functional fragment or variant thereof. In some embodiments, the MUC1* binding domain comprises an IgG1, IgG2, IgG3, or IgG4 domain. In some embodiments, the MUC1* binding domain comprises an IgG1 domain. In some embodiments, the MUC1* binding domain comprises an IgG2 domain. In some embodiments, the MUC1* binding domain comprises an IgG3 domain. In some embodiments, the MUC1* binding domain comprises an IgG4 domain.

[0073] In some embodiments, an antibody, or functional fragment or variant thereof, that specifically binds to MUC1* comprises an anti-MUC1* heavy chain and an anti-MUC1* light chain.

[0074] In some embodiments, the anti-MUC1* heavy chain comprises an anti-MUC1* heavy chain variable domain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, the anti-MUC1* light chain comprises an anti-MUC1* light chain variable domain. In some embodiments, the anti-MUC1* light chain variable domain comprises the variable domain of a kappa or lambda light chain.

[0075] In some embodiments, the anti-MUC1* heavy chain variable domain comprises a variable domain of an IgG1 heavy chain and the anti-MUC1* light chain variable domain comprises a variable domain of a kappa or lambda light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises a variable domain of an IgG2 heavy chain and the anti-MUC1* light chain variable domain comprises a variable domain of a kappa or lambda light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises a variable domain of an IgG3 heavy chain and the anti-MUC1* light chain variable domain comprises a variable domain of a kappa or lambda light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises a variable domain of an IgG4 heavy chain and the anti-MUC1* light chain variable domain comprises a variable domain of a kappa or lambda light chain.

[0076] In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG1 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a kappa light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG2 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a kappa light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG3 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a kappa light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG4 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a kappa light chain.

[0077] In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG1 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a lambda light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG2 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a lambda light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG3 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a lambda light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG4 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a lambda light chain.

[0078] In some embodiments, an antibody, or functional fragment or variant thereof, that specifically binds to MUC1* comprises a single chain variable fragment (scFv) or an antigen-binding fragment (Fab). In some embodiments, an antibody, or functional fragment or variant thereof, that specifically binds to MUC1* comprises a single chain variable fragment. In some embodiments, an antibody, or functional fragment or variant thereof, that specifically binds to MUC1* comprises an antigen-binding fragment (Fab).

[0079] In some embodiments, the anti-MUC1* heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the anti-MUC1* heavy chain variable domain comprise the amino acid sequences set forth in: HC-CDR1: SEQ ID NO: 1 or 4, HC-CDR2: SEQ ID NO: 2 or 5, and HC-CDR3: SEQ ID NO: 3 or 6, and the CDRs comprise 0 to 2 amino acid modifications (e.g., 0 or 1 amino acid modifications) in at least one of HC-CDR1, HC-CDR2, or HC-CDR3.

[0080] In some embodiments, the anti-MUC1* light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the anti-MUC1* light chain variable domain comprise the amino acid sequences set forth in LC-CDR1: SEQ ID NO: 13 or 16, LC-CDR2: SEQ ID NO: 14 or 17, and LC-CDR3: SEQ ID NO: 15 or 18, and the CDRs comprise 0 to 2 amino acid modifications (e.g., 0 or 1 amino acid modifications) in at least one of LC-CDR1, LC-CDR2, or LC-CDR3.

[0081] In some embodiments, the anti-MUC1* heavy chain comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 38 or 44. In some embodiments, the anti-MUC1* light chain comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 41 or 47.

[0082] [Table 1]

[0083] [Table 2-1]

[0084] [Table 2-2]

[0085] [Table 3]

[0086] CD3-binding domain In some embodiments, disclosed herein is an antibody comprising an anti-CD3 binding domain. In some embodiments, the CD3 binding domain comprises an antibody or an antigen-binding fragment or variant thereof. In some embodiments, the antibody or an antigen-binding fragment or variant thereof is a monoclonal antibody. In some embodiments, the antibody or an antigen-binding fragment or variant thereof is a human antibody, a murine antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the CD3 binding domain comprises a monovalent Fab, a bivalent Fab'2, a single chain variable fragment (scFv), or a functional fragment or variant thereof.

[0087] In some embodiments, the CD3 binding domain comprises an IgG1, IgG2, IgG3, or IgG4 domain. In some embodiments, the CD3 binding domain comprises an IgG1 domain. In some embodiments, the CD3 binding domain comprises an IgG2 domain. In some embodiments, the CD3 binding domain comprises an IgG3 domain. In some embodiments, the CD3 binding domain comprises an IgG4 domain.

[0088] In some embodiments, the antibody, or functional fragment or variant thereof, that specifically binds to CD3 comprises an anti-CD3 heavy chain and an anti-CD3 light chain.

[0089] In some embodiments, the anti-CD3 heavy chain comprises an anti-CD3 heavy chain variable domain. In some embodiments, the anti-CD3 heavy chain variable domain comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, the anti-CD3 light chain comprises an anti-CD3 light chain variable domain. In some embodiments, the anti-CD3 light chain variable domain comprises a variable domain of a kappa or lambda light chain. In some embodiments, the anti-CD3 heavy chain variable domain comprises a variable domain of an IgG1 heavy chain and the anti-CD3 light chain variable domain comprises a variable domain of a kappa or lambda light chain. In some embodiments, the anti-CD3 heavy chain variable domain comprises a variable domain of an IgG2 heavy chain and the anti-CD3 light chain variable domain comprises a variable domain of a kappa or lambda light chain. In some embodiments, the anti-CD3 heavy chain variable domain comprises a variable domain of an IgG3 heavy chain and the anti-CD3 light chain variable domain comprises a variable domain of a kappa or lambda light chain. In some embodiments, the anti-CD3 heavy chain variable domain comprises a variable domain of an IgG4 heavy chain and the anti-CD3 light chain variable domain comprises a variable domain of a kappa or lambda light chain.

[0090] In some embodiments, an antibody, or functional fragment or variant thereof, that specifically binds to CD3 comprises a single chain variable fragment (scFv) or an antigen-binding fragment (Fab). In some embodiments, an antibody, or functional fragment or variant thereof, that specifically binds to CD3 comprises a single chain variable fragment (scFv). In some embodiments, an antibody, or functional fragment or variant thereof, that specifically binds to CD3 comprises an antigen-binding fragment (Fab).

[0091] In some embodiments, the anti-CD3 heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the anti-CD3 heavy chain variable domain comprise the amino acid sequences set forth in HC-CDR1: SEQ ID NO: 7 or 10, HC-CDR2: SEQ ID NO: 8 or 11, and HC-CDR3: SEQ ID NO: 9 or 12, and the CDRs comprise 0 to 2 amino acid modifications (e.g., 0 or 1 amino acid modifications) in at least one of HC-CDR1, HC-CDR2, or HC-CDR3.

[0092] In some embodiments, the anti-CD3 light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, and the LC-CDR1, LC-CDR2, and LC-CDR3 of the anti-CD3 light chain variable domain comprise the amino acid sequences set forth in LC-CDR1: SEQ ID NO: 19 or 22, LC-CDR2: SEQ ID NO: 20 or 23, and LC-CDR3: SEQ ID NO: 21 or 24, and the CDRs comprise 0 to 2 amino acid modifications (e.g., 0 or 1 amino acid modifications) in at least one of LC-CDR1, LC-CDR2, or LC-CDR3.

[0093] In some embodiments, the anti-CD3 heavy chain comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 26 or 32.

[0094] In some embodiments, the anti-CD3 heavy chain comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 30 or 33.

[0095] In some embodiments, the anti-CD3 heavy chain comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the amino acid sequences in Table 5.

[0096] In some embodiments, the anti-CD3 light chain comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the amino acid sequences in Table 5.

[0097] [Table 4]

[0098] [Table 5-1]

[0099] [Table 5-2]

[0100] [Table 5-3]

[0101] [Table 6]

[0102] In some embodiments, the antibody, or functional fragment or functional variant thereof, that specifically binds to MUC1* comprises a Fab and the antibody, or functional fragment or functional variant thereof, that specifically binds to CD3 comprises an scFv.

[0103] In some embodiments, the anti-MUC1* heavy chain comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 38 or 44, and the anti-MUC1* light chain comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 41 or 47. and the scFv comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 131 or 132.

[0104] In some embodiments, the antibody, or functional fragment or variant thereof, that specifically binds to MUC1* comprises an scFv and the antibody, or functional fragment or variant thereof, that specifically binds to CD3 comprises a Fab.

[0105] In some embodiments, the anti-CD3 heavy chain comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:26, 27, 32, or 33, and the anti-CD3 light chain comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:29, 30, 35, or 36. and the scFv comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 129 or 130.

[0106] In some embodiments, the antibody comprises at least three CDRs of an anti-MUC1* binding domain selected from any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 13, 14, 15, 16, 17, or 18, and 0-2 amino acid modifications thereof (e.g., 0-1 amino acid modifications), and at least three CDRs of an anti-CD3 binding domain selected from any one of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 19, 20, 21, 22, 23, or 24, and 0-2 amino acid modifications thereof (e.g., 0-1 amino acid modifications).

[0107] In some embodiments, the antibody, or functional fragment or functional variant thereof, that specifically binds to MUC1* comprises an scFv and the antibody, or functional fragment or functional variant thereof, that binds to CD3 comprises an scFv.

[0108] In some embodiments, the anti-MUC1* scFv comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 129 or 130. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 131 or 132.

[0109] [Table 7-1]

[0110] [Table 7-2]

[0111] [Table 7-3]

[0112] [Table 7-4]

[0113] [Table 7-5]

[0114] [Table 7-6]

[0115] [Table 7-7]

[0116]

Table 7-8

[0117]

Table 7-9

[0118]

Table 7-10

[0119]

Table 7-11

[0120]

Table 7-12

[0121]

Table 7-13

[0122]

Table 7-14

[0123]

Table 7-15

[0124]

Table 7-16

[0125]

Table 7-17

[0126]

Table 7-18

[0127]

Table 7-19

[0128]

Table 7-20

[0129]

Table 7-21

[0130]

Table 7-22

[0131]

Table 7-23

[0132]

Table 7-24

[0133]

Table 7-25

[0134]

Table 7-26

[0135]

Table 7-27

[0136]

Table 7-28

[0137]

Table 7-29

[0138]

Table 7-30

[0139]

Table 7-31

[0140]

Table 7-32

[0141]

Table 7-33

[0142]

Table 7-34

[0143]

Table 7-35

[0144]

Table 7-36

[0145]

Table 7-37

[0146]

Table 7-38

[0147] [Table 7-39]

[0148] [Table 7-40]

[0149] [Table 7-41]

[0150] In some embodiments, the antibody further comprises a fragment crystallizable (Fc) region. In some embodiments, the Fc region comprises an IgG CH2 domain and an IgG CH3 domain. In some embodiments, the Fc region comprises a heterodimeric Fc region. In some embodiments, the heterodimeric Fc region comprises an (e.g., human) IgG1, IgG2, IgG3, or IgG4 domain. In some embodiments, the heterodimeric Fc region comprises an (e.g., human) IgG1 domain. In some embodiments, the heterodimeric Fc region comprises an (e.g., human) IgG2 domain. In some embodiments, the heterodimeric Fc region comprises an (e.g., human) IgG3 domain. In some embodiments, the heterodimeric Fc region comprises an (e.g., human) IgG4 domain.

[0151] In some embodiments, the heterodimeric Fc region comprises a knob chain and a hole chain, and has a knob-into-hole (KIH) structure (Spiess et al. Molecular Immunology 67, 95-106 (2015) format. In some embodiments, the knob chain comprises a (e.g., human) IgG1, IgG2, IgG3, or IgG4 domain. In some embodiments, the knob chain comprises a (e.g., human) IgG1 domain. In some embodiments, the knob chain comprises a (e.g., human) IgG2 domain. In some embodiments, the knob chain comprises a (e.g., human) IgG3 domain. In some embodiments, the knob chain comprises a (e.g., human) IgG4 domain. In some embodiments, the hole chain comprises a (e.g., human) IgG1, IgG2, IgG3, or IgG4 domain. In some embodiments, the hole chain comprises a (e.g., human) IgG1 domain. In some embodiments, the hole chain comprises a (e.g., human) IgG2 domain. In some embodiments, the hole chain comprises a (e.g., human) IgG3 domain. In some embodiments, the hole chain comprises a (e.g., human) IgG4 domain.

[0152] In some embodiments, the target cell is a cancer cell, hi some embodiments, the cancer is breast cancer, colon cancer, prostate cancer, pancreatic cancer, or lung cancer.

[0153] In some embodiments, the antibody binds to cancer cells that express MUC1* on their surface.

[0154] As an example of how the antibodies of the invention can be incorporated into a bispecific antibody, a bispecific antibody was constructed using a knob-in-hole, also known as KIH (Spiess et al. Molecular Immunology 67, 95-106 (2015)). In this example, the first arm of the antibody is the humanized anti-MUC1* antibody 20A10, also known as hu20A10, with 14616 human framework regions, and the second arm of the antibody is either the anti-CD3 antibody OKT3 or 12F6, both of which bind to the same epitope on human T cells. The resulting bispecific antibodies are referred to as 20A10-OKT3-BiTE and 20A10-12F6-BiTE. In a proof of function, the bispecific antibody is added at various concentrations to cultured cells in which both human T cells and MUC1* positive cancer cells are present. In one case, the cancer cells were T47D breast cancer cells, and in the other case, MUC1*-negative line HCT-116 colon cancer cells were transduced to express MUC1*, designated HCT-MUC1*. As seen in the photographs shown in Figures 1A-1L, 2A-2L, 3A-3L, and 4A-4L, the addition of either bispecific antibody mediated binding of T cells to MUC1*-positive cancer cells, as evidenced by bispecific dose-dependent cell clustering. Two control experiments were performed. In one control, no bispecific antibody was added. In another control, only MUC1* cancer cells are present. No clustering is observed. In another control, bispecific antibody is added to MUC1*-positive cancer cells, but no T cells are present. Additionally, cancer cell killing was quantified. The cytotoxicity of an anti-MUC1* targeting bispecific antibody, 20A10, which binds to the extracellular domain of MUC1* on the surface of tumor cells, and an anti-CD3 antibody, termed OKT3, was measured using an LDH cytotoxicity assay. A cartoon depicting how the LDH cytotoxicity assay works is shown (Figure 1M), with higher readings at A490 indicating higher cell killing rates. Figure 1N shows a graph of cell killing as a function of concentration of 20A10-OKT3, an anti-MUC1* / anti-CD3 bispecific antibody.As can be seen in the graph, 20A10 incorporated into a bispecific antibody, where the second arm is an antibody or antibody fragment that binds to CD3 or other surface molecules on T cells, potently kills MUC1*-positive tumor cells. In addition to analyzing the target cancer cells, T cells were also analyzed. As is well known, activated CD8-positive cytotoxic T cells secrete interferon gamma (IFN-g) upon activation and primed killing. Secretion of IFN-g from T cells into conditioned medium was measured by ELISA assay (Figure 1O). As can be seen in the figure, half-maximal secretion of IFN-g is achieved at a bispecific concentration as low as 12.3 ng / mL. Activated T cells also secrete TNF-α into conditioned medium. ELISA measurement of TNF-a also shows half-maximal secretion at a very low concentration of 12.3 ng / mL of 20A10-CD3 bispecific antibody (Figure 1P).

[0155] In yet another example, an anti-MUC1* / anti-CD3 bispecific antibody is constructed using 20A10 to bind to the extracellular domain of MUC1*-positive cancer cells and anti-CD3 antibody 12F6 to bind to T cells. As seen in the photographs shown in Figures 2A-2L, the addition of either bispecific antibody mediated the binding of T cells to MUC1*-positive cancer cells as evidenced by the bispecific's dose-dependent cell clustering. Two control experiments were performed. In one control, no bispecific antibody is added. In another control, only MUC1* cancer cells are present. No clustering is observed. In another control, the bispecific antibody is added to MUC1*-positive cancer cells but no T cells are present. Furthermore, the killing of cancer cells was quantified. The cytotoxicity of the anti-MUC1*-targeting bispecific antibody, where 20A10 binds to the extracellular domain of MUC1* on the tumor cell surface, and the anti-CD3 antibody, designated 12F6, was measured using an LDH cytotoxicity assay. A cartoon depicting how the LDH cell cytotoxicity assay works is shown (Figure 2M), indicating that the higher the readings at A490, the higher the cell killing rate. Figure 2N shows a graph of cell killing as a function of concentration of 20A10-12F6, an anti-MUC1* / anti-CD3 bispecific antibody. As can be seen in the graph, 20A10 incorporated into a bispecific antibody, where the second arm is an antibody or antibody fragment that binds to CD3 or other surface molecules on T cells, potently kills MUC1*-positive tumor cells. In addition to analyzing the target cancer cells, T cells were also analyzed. As is well known, activated CD8-positive cytotoxic T cells secrete interferon gamma (IFN-g) when activated and primed to kill. Secretion of IFN-g from T cells into conditioned medium was measured by ELISA assay (Figure 2O). As can be seen in the figure, half-maximal secretion of IFN-g is achieved at bispecific concentrations as low as 50 ng / mL. Activated T cells also secrete TNF-α into the conditioned medium, and ELISA measurements of TNF-α show half-maximal secretion at a very low concentration of 200 ng / mL of the 20A10-CD3 bispecific antibody (Figure 2P).The difference in efficacy between 20A10 in combination with 12F6 and 20A10 in combination with OKT3 indicates that anti-MUC1* / anti-T cell potency is modulated by the affinity and specificity of each of the two antibodies.

[0156] In yet another example, anti-MUC1* antibody 20A10 is paired with an anti-CD3 antibody fragment to test for killing of HCT-116 colon cancer cells, a MUC1*-negative line transduced to express MUC1*, referred to as HCT-MUC1*. In one case, the antibody fragment for binding to T cells is OKT3. In another case, the anti-CD3 antibody is 12F6. As seen in the photographs in Figures 3A-3L (OKT3) and Figures 4A-4L (12F6), addition of either bispecific antibody mediated binding of T cells to MUC1*-positive cancer cells, as evidenced by bispecific dose-dependent cell clustering. Two control experiments were performed. In one control, no bispecific antibody is added, but both T cells and MUC1* cancer cells are present. No clustering is observed. In another control, bispecific antibody is added to MUC1*-positive cancer cells, but no T cells are present.

[0157] Antibody-drug conjugates Antibody-drug conjugates (ADCs) allow for targeted delivery of therapeutic agents to specific cells and / or tissues. In certain embodiments, an ADC comprises an agent (e.g., a therapeutic agent) that can inhibit topoisomerase (e.g., topoisomerase I (TopoI)) or inhibit tubulin production.

[0158] In another aspect, provided herein are conjugates, e.g., antibody-drug conjugates. The ADCs comprise a therapeutic agent (represented as X), a linking moiety (represented as L), a binding moiety (represented as R), and an antibody (

[0159] [ka] In some embodiments, intracellular cleavage of the L linker results in a conjugate moiety (represented as Z) to

[0160] [ka] This allows for separation of therapeutic agent X from the target cell, thereby facilitating cellular uptake or tissue retention of therapeutic agent X.

[0161] In one embodiment, the ADC has the formula (I): [Ab]-[ZLRX] y wherein X is a moiety derived from a compound capable of inhibiting topoisomerase I or a compound capable of inhibiting tubulin formation, R is a binding moiety, and L is a dipeptide, tripeptide or tetrapeptide binding moiety having Z attached to the N-terminus and R attached to the C-terminus, [Ab] is an antibody comprising an anti-MUC1* binding domain comprising three heavy chain (HC) complementarity determining regions (CDRs): MUC1* HC-CDR1, MUC1* HC-CDR2, and MUC1* HC-CDR3, wherein MUC1* HC-CDR1, MUC1* HC-CDR2, and MUC1* HC-CDR3 of the MUC1* binding domain comprise an amino acid sequence selected from those set out in Table 1, and wherein the MUC1* binding domain comprises three light chain (LC) complementarity determining regions (CDRs): MUC1* LC-CDR1, MUC1* LC-CDR2, and MUC1* and wherein MUC1* LC-CDR1, MUC1* LC-CDR2, and MUC1* LC-CDR3 of the MUC1* binding domain comprise an amino acid sequence selected from those shown in Table 1, Z is a conjugating moiety capable of forming a covalent bond with the sulfur atom of a cysteine ​​residue, and y is an integer from 1 to 10.

[0162] The ADC has the structure provided below:

[0163] [ka] wherein n is 1 to 10.

[0164] The ADC has the structure provided below:

[0165] [ka] wherein n is 1 to 10.

[0166] The efficacy of an ADC depends in part on the drug-to-antibody ratio, or DAR. Essentially, the more toxin conjugated to the antibody, the more cell killing there is. However, conjugating too much toxin to the antibody can destabilize the antibody or sterically interfere with the interaction between the antibody and the target antigen. Hydrophobic interaction chromatography (HIC) is a bioanalytical technique used to determine the drug-to-antibody ratio (DAR) of antibody-drug conjugates (ADCs). A HIC column on a high pressure liquid chromatography (HPLC) system is used for the analysis and characterization of ADCs with a salt gradient buffer. HIC separates proteins according to differences in their surface hydrophobicity, and the more drug that is conjugated to the antibody, the longer the retention time. Figures 30-35 show HIC chromatograms for a batch of MNC2 and a batch of MN20A10 conjugated to several toxic payloads, as well as the corresponding calculated DARs for each.

[0167] In some embodiments, the DAR of the conjugates provided herein ranges from 1 to 20. In some embodiments, the DAR of the conjugates provided herein ranges from 1 to 15. In some embodiments, the DAR of the conjugates provided herein ranges from 1 to 10. In some embodiments, the DAR of the conjugates provided herein ranges from 1 to 8. In some embodiments, the DAR of the conjugates provided herein ranges from 1 to 7. In some embodiments, the DAR of the conjugates provided herein ranges from 1 to 6. In some embodiments, the DAR of the conjugates provided herein ranges from 1 to 5. In some embodiments, the DAR of the conjugates provided herein ranges from 1 to 4.

[0168] In certain embodiments, the DAR of the conjugates provided herein is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12. In some embodiments, the DAR of the conjugates provided herein is about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. In some embodiments, the DAR of the conjugates provided herein is about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0169] In some embodiments, the DAR of the conjugates provided herein is about 1. In some embodiments, the DAR of the conjugates provided herein is about 2. In some embodiments, the DAR of the conjugates provided herein is about 3. In some embodiments, the DAR of the conjugates provided herein is about 4. In some embodiments, the DAR of the conjugates provided herein is about 3.8. In some embodiments, the DAR of the conjugates provided herein is about 5. In some embodiments, the DAR of the conjugates provided herein is about 6. In some embodiments, the DAR of the conjugates provided herein is about 7. In some embodiments, the DAR of the conjugates provided herein is about 8.

[0170] In certain embodiments, fewer than the theoretical maximum units are conjugated to a polypeptide, e.g., an antibody, during a conjugation reaction.

[0171] In some embodiments, the amino acid that binds to the unit is in the heavy chain of the antibody. In some embodiments, the amino acid that binds to the unit is in the light chain of the antibody. In some embodiments, the amino acid that binds to the unit is in the hinge region of the antibody. In some embodiments, the amino acid that binds to the unit is in the Fc region of the antibody. In some embodiments, the amino acid that binds to the unit is in the constant region of the antibody (e.g., CH1, CH2, or CH3 of the heavy chain, or CH1 of the light chain). In yet other embodiments, the amino acid that binds to the unit or drug unit is in the VH framework region of the antibody. In yet other embodiments, the amino acid that binds to the unit is in the VL framework region of the antibody.

[0172] It should be understood that the preparation of the conjugates described herein may result in a mixture of conjugates with one or more units bound to the polypeptide (i.e., heterogeneous), for example, antibody, distributed. Individual conjugate molecules can be identified in the mixture by mass spectrometry and separated by HPLC, for example, hydrophobic interaction chromatography. In some embodiments, homogeneous conjugates with a single DAR (loading) value can be isolated from the conjugate mixture by electrophoresis or chromatography.

[0173] The present disclosure provides an antibody-drug conjugate (ADC) comprising a monoclonal antibody against MUC1* or an antigen-binding fragment thereof conjugated to a cytotoxin. As used herein, the term "antibody-drug conjugate" refers to a compound comprising a monoclonal antibody (mAb) linked to a cytotoxic agent (generally a small molecule drug with high systemic toxicity) via a chemical linker. In some embodiments, the ADC may comprise a small molecule cytotoxin chemically modified to include a linker. The linker is then used to conjugate the cytotoxin to the antibody or its antigen-binding fragment. Upon binding to a target antigen at the surface of a cell, the ADC is internalized and transported to the lysosome, where the cytotoxin is released by proteolysis of the cleavable linker (e.g., by cathepsin B found in lysosomes) or by proteolysis of the antibody if the cytotoxin is attached to the cytotoxin via a non-cleavable linker. The cytotoxin then travels from the lysosome to the cytosol or the nucleus, where it can then bind to a target, depending on its mechanism of action. The antibody-drug conjugates described herein may comprise whole antibodies or antibody fragments. The parent antibody may be murine, rabbit, human, humanized, camelid or other species.

[0174] ADCs may include antigen-binding fragments of antibodies. The terms "antibody fragment," "antigen-binding fragment," "functional fragment of an antibody," and "antigen-binding portion" are used interchangeably herein and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., Nat. Biotech., 23(9):1 126-1129 (2005)). An antibody fragment may include, for example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. Examples of antibody fragments include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; and (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of the two domains (i.e., VL and VH) of an Fv fragment linked by a synthetic linker which allows the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Osbourn et al., J. Immunol. 1999, 11:1311-1315 (1999)). al., Nat. Biotechnol., 16:778 (1998)), and (v) diabodies that are dimers of polypeptide chains, where each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to permit pairing between the VH and VL on the same polypeptide chain, thereby promoting pairing between complementary domains on different VH-VL polypeptide chains to generate a dimeric molecule having two functional antigen-binding sites.

[0175] The terms cytotoxin and cytotoxic agent refer to any molecule that inhibits or prevents the function of a cell and / or causes destruction of a cell (cell death) and / or exerts an anti-proliferative effect. It will be understood that the cytotoxin or cytotoxic agent of an ADC is also referred to in the art as the "payload" of the ADC. Several classes of cytotoxic agents are known in the art to have potential utility in ADC molecules and can be used in the ADCs described herein. Such classes of cytotoxic agents include, for example, anti-microtubule agents (e.g., auristatins and maytansinoids), pyrrolobenzodiazepines (PBDs), RNA polymerase II inhibitors (e.g., amatoxins), and DNA alkylating agents (e.g., indolinobenzodiazepine pseudodimers). Examples of specific cytotoxic agents that may be used in the ADCs described herein include, but are not limited to, amanitin, auristatin, calicheamicin, daunomycin, doxorubicin, duocarmycin, dolastatin, enediyne, lexitropsin, taxane, puromycin, maytansinoid, vinca alkaloid, tubulysin, and pyrrolobenzodiazepine (PBD). More specifically, the cytotoxic agent may be, for example, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholinodoxorubicin, rhizoxin, cyanomorpholinodoxorubicin, dolastatin 10, echinomycin, combretastatin, calicheamicin, maytansine, DM1, DM4, vinblastine, methotrexate, netropsin, or a derivative or analog thereof.

[0176] Auristatins are a class of highly potent antimitotic agents that have shown substantial preclinical activity and are well tolerated. Examples of auristatins that may be used in conjunction with the ADCs described herein include, but are not limited to, monomethylauristatin E (MMAE) and the related molecule monomethylauristatin F (MMAF).

[0177] In one embodiment, the cytotoxic agent can be a pyrrolobenzodiazepine (PBD) or a PBD derivative. PBDs migrate to the cell nucleus, where they crosslink DNA, block replication during mitosis, and damage DNA by inducing single-strand breaks, which then leads to apoptosis. Some PBDs also have the ability to recognize and bind to specific sequences of DNA.

[0178] The monoclonal antibodies described herein are conjugated to at least one cytotoxin molecule, however, the anti-MUC1* monoclonal antibodies may be conjugated to any suitable number of cytotoxin molecules (e.g., 1, 2, 3, 4 or more cytotoxin molecules) to achieve the desired therapeutic effect.

[0179] The present disclosure also provides a composition comprising the above-mentioned antibody or antibody-drug conjugate and a pharma- ceutically acceptable (e.g., physiologically acceptable) carrier. Any suitable carrier known in the art can be used within the context of the present invention. The choice of carrier is determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition may optionally be sterile. The composition can be produced according to conventional techniques, for example, as described in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2001).

[0180] The composition desirably comprises an amount of the antibody or antibody-drug conjugate effective to treat or prevent MUC1*-expressing cancer.Thus, the disclosure provides a method of killing MUC1* positive cells, comprising contacting a cell expressing MUC1* with an antibody or antibody-drug conjugate described herein, or a composition comprising an antibody or ADC described herein, whereby the antibody or antibody-drug conjugate binds to MUC1* on the cell and kills the cell.

[0181] The disclosure also provides the use of an antibody or ADC, or a composition comprising an antibody or ADC, described herein in the manufacture of a medicament for treating a MUC1*-positive cancer.

[0182] As demonstrated herein, MUC1* is expressed in a variety of cancer types. Accordingly, the present disclosure provides a method of killing such cancer cells, comprising contacting a cancer cell expressing MUC1* with a composition comprising an antibody-drug conjugate described herein, or an ADC described herein, whereby the antibody-drug conjugate binds to MUC1* on the cell and kills the cell.

[0183] An antibody-drug conjugate described herein, or a composition comprising an antibody-drug conjugate, may be contacted with a population of cells expressing MUC1* ex vivo, in vivo or in vitro, preferably in vivo.

[0184] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is therapeutic. That is, the effect partially or completely treats the disease and / or the adverse symptoms resulting from the disease. To this end, the method of the present invention includes administering a "therapeutically effective amount" of an antibody or ADC, or a composition comprising an antibody or ADC and a pharma- ceutically acceptable carrier. A "therapeutically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, weight of the individual, and the ability of the antibody or ADC to elicit a desired response in the individual. For example, a therapeutically effective amount of the ADC of the present invention is an amount that binds to MUC1* on MUC1*-positive cells and destroys them.

[0185] Alternatively, the pharmacological and / or physiological effect may be a prophylactic effect, i.e., an effect that completely or partially prevents a disease or its symptoms. In this regard, the methods of the invention comprise administering a "prophylactically effective amount" of an ADC or a composition comprising the ADC to a mammal susceptible to a MUC1*-expressing cancer. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (e.g., prevention of disease onset).

[0186] Therapeutic or prophylactic efficacy can be monitored by periodic evaluation of treated patients. In one embodiment, the ADCs described herein inhibit or suppress proliferation of MUC1*-expressing cells by at least about 10% (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%). Cell proliferation can be measured using any suitable method known in the art, such as measuring the incorporation of labeled nucleosides (e.g., 3H-thymidine or bromodeoxyuridine Brd(U)) into genomic DNA (see, e.g., Madhavan, HN, J. Stem Cells Regen. Med., 3(1):12-14 (2007)).

[0187] The antibodies or ADCs described herein, or compositions comprising antibody ADCs, can be administered to a mammal (e.g., a human) using standard administration techniques, including, for example, intravenous, intraperitoneal, subcutaneous, etc. More preferably, the antibodies or ADCs, or compositions containing them, are administered to the mammal by intravenous injection.

[0188] The antibody or ADC described herein, or a composition comprising the antibody or ADC, may be administered with one or more additional therapeutic agents that can be co-administered to a mammal. As used herein, the term "co-administration" refers to administration of one or more additional therapeutic agents and an antibody or ADC described herein, or an antibody or ADC-containing composition, close enough in time that the antibody or ADC can enhance the effect of the one or more additional therapeutic agents, or conversely, the one or more additional therapeutic agents can enhance the effect of the antibody or ADC. In this regard, the antibody or ADC or a composition containing the same may be administered first, and the one or more additional therapeutic agents may be administered second, or vice versa. For example, the antibody or ADC or a composition containing the same may be administered in combination with other agents (e.g., as an adjuvant) for the treatment or prevention of MUC1*-positive cancer. In this regard, the antibody or ADC or antibody- or ADC-containing composition can be used in combination with at least one other anti-cancer agent, including, for example, any suitable chemotherapeutic agent known in the art, ionizing radiation, small molecule anti-cancer agents, cancer vaccines, biological therapies (e.g., other monoclonal antibodies, cancer-killing viruses, gene therapy, adoptive T-cell transfer), and / or surgery.

[0189] As an example of how the antibodies of the present invention can be incorporated into ADCs, antibody drug conjugates, for the treatment of MUC1*-positive cancers, a toxin was attached to the anti-MUC1* antibody MNC2. In this particular example, the toxin is MMAE, i.e., monomethyl auristatin, but there are a variety of toxins and functional linkers known to those skilled in the art that facilitate the killing of target cells, particularly cancer cells, by ADCs. In this example, MNC2 was deglycosylated and then reacted with a linker that facilitates the covalent attachment of MMAE, referred to herein as MNC2-ADC. MNC2-ADC was incubated with target cells at various concentrations and for various times. The cells were then photographed and cell viability was measured using an indicator of cell death called PrestoBlue™ (Thermo Fisher Scientific, Waltham, MA). MNC2-ADC incubated with HCT, a MUC1 and MUC1*-negative cell line, did not induce cell death at any concentration (Figures 5A-5F). However, when HCT cells were transduced to express MUC1* to generate HCT-MUC1*, MNC2-ADC induced cell death in a dose-dependent manner (Figures 6A-6G). A graph of the measured loss of cell viability is shown in Figure 9B. Similarly, MNC2-ADC did not induce cell death in K562 cells, which are MUC1*-negative cells (Figures 7A-7G). Again, when cells were transduced to express MUC1* to generate K562-MUC1*, MNC2-ADC was able to induce cell death in a concentration-dependent manner (Figures 8A-8G). A graph of the measured loss of cell viability is shown in Figure 9A.

[0190] T47D cells are a naturally occurring breast cancer cell line that expresses both full-length MUC1, which MNC2 does not bind, and MUC1*, which MNC2 does bind. T47D-MUC1* is a cell line in which T47D cells were transduced to express more MUC1*. MNC2-ADC was incubated with T47D-WT cells (Figures 10A-10G) or T47D-MUC1* cells (Figures 11A-11G). As can be seen in the graph of measured cell viability (Figure 12), only T47D-MUC1* cells died at the highest concentration tested. Another experiment was performed in which the concentration of added MNC2-ADC was significantly increased. In this case, T47D-WT cells were killed by MNC2-ADC at 1000 nM (Figures 13A-13J), and T47D-MUC1* cells were killed at 10 nM, 39 nM, 100 nM, 393 nM, and 1000 nM (Figures 14A-14J). Graphs measuring cell death induced by MNC2-ADC in T47D and T47D-MUC1* are shown in Figure 15A. These experiments are shown by way of example only. For an ADC to function, engagement of the ADC with the target cell must induce receptor internalization, and this set of examples demonstrates that. The toxin attached to the anti-MUC1* antibody may be one of several known to those skilled in the art. Similarly, MNC2 may be human or humanized.

[0191] However, optimization of the toxin coupling chemistry significantly improved the results of MNC2-ADC. In this next experiment, a valine-citrulline p-aminobenzylcarbamate (VC-PAB) linker was used to link the deglycosylated antibody to the toxin. The linkers were individually conjugated to MMAE at one end, then reacted at the other end with maleimide, which was then conjugated to the antibody after reduction by alkylation. MMAE inhibits cell division by blocking tubulin polymerization. The linker, which is stable outside the cell, is cleaved by cathepsin B, allowing the ADC to be internalized, which activates MMAE. This optimized MNC2-ADC was incubated with either T47D wild type cells (-WT) (Figures 16A and 16C) or T47D cells transduced to express more MUC1* (T47D-MUC1*) (Figures 16B and 16D) over concentrations ranging from 0.1 ng / mL to 500 ng / mL. In some cases, MNC2-ADC was incubated with 5,000 target MUC1* positive cells per well of a 96-well plate for 72 hours (Figures 16A-16B). Alternatively, MNC2-ADC was incubated with target MUC1* positive cells for only 16 hours, after which the medium was changed and the cells were left in culture for an additional 54 hours (Figures 16C-16D). For comparison, the well-known anti-cancer drug Taxol was added to T47D-wt or T47D-MUC1* cells for 72 hours (Figures 16E-16F). The viability of the 72-hour co-cultures was measured using an indicator of cell death called PrestoBlue™ (Thermo Fisher Scientific, Waltham, MA) (FIG. 17). As can be seen, cells with higher antigen density died faster at lower MNC2-ADC concentrations. As much as 90% of the MUC1* high cells died at 100 nM MNC2-ADC concentration over the 72-hour incubation.

[0192] As another example of how the antibodies of the present invention can be incorporated into ADCs, antibody drug conjugates, for the treatment of MUC1*-positive cancers, a toxin was attached to another anti-MUC1* antibody, 20A10. Magnified images of remaining cancer cells were taken after 72 hours of co-culture with 20A10-ADC (Figures 18A-18F). In this particular example, the toxin is MMAE, i.e., monomethyl auristatin, but there are a variety of toxins and functional linkers known to those skilled in the art that facilitate the killing of target cells, particularly cancer cells, by ADCs. 20A10 was deglycosylated and then reacted with a linker that facilitates the covalent attachment of MMAE, referred to herein as 20A10-ADC or 20A10-MMAE. 20A10-ADC was incubated with either T47D wild type cells (-WT) (Figures 18A and 18C) or T47D cells transduced to express more MUC1* (T47D-MUC1*) (Figures 18B and 18D) over concentrations ranging from 0.1 ng / mL to 500 ng / mL. In some cases, 20A10-ADC was incubated with the target MUC1* positive cells for the entire 72 hour assay (Figures 18A-18B). Alternatively, 20A10-ADC was incubated with the target MUC1* positive cells for only 16 hours, after which the medium was changed and the cells were left in culture for an additional 54 hours (Figures 18C-18D). For comparison, the well-known anti-cancer drug Taxol was added to T47D-wt or T47D-MUC1* cells for 72 hours (Figures 18E-18F). The viability of the 72-hour co-cultures was measured using an indicator of cell death called PrestoBlue™ (Thermo Fisher Scientific, Waltham, MA) (FIG. 19). As can be seen, the higher the antigen density, the more cancer cells are killed. Even after 16 hours of incubation, over 80% of the high MUC1* cells were killed, and after 72 hours of incubation, 100% were killed at concentrations below 10 nM. Cells with low MUC1* expression required higher 20A10-ADC concentrations and longer incubation periods to result in 100% cell killing.

[0193] The graph in Figure 20A plots the IC50s from these experiments, and the data is tabulated in Figure 20B.

[0194] These experiments demonstrate that, using optimized conjugation chemistries and new and improved toxins and linkers, anti-MUC1* antibodies can be successfully incorporated into a number of ADC formats.

[0195] One in vitro method to characterize each ADC is to measure the ability of the antibody to bind to the target antigen before and after the toxin is conjugated to the antibody. The specificity of the antibody may be compromised by the process of chemically conjugating some toxins to the antibody. This phenomenon is due to the inherent stability or instability of each antibody rather than due to any element of the conjugation process. In many cases, the payload is attached to the antibody via binding to a free thiol generated by cleaving or reducing a disulfide bond. The disulfide bond holds the two heavy chains together and supports the structure of the variable region, the antibody recognition unit. A significant challenge is to reduce enough disulfide bonds to allow the attachment of multiple toxins without destroying the disulfide bonds that maintain the critical antibody structure. Surprisingly, MNC2 is a very stable and well-behaved antibody that allows the attachment of many toxins without compromising the antibody structure or changing the antibody's ability to recognize the target antigen. The challenges faced when attaching payloads to the antibody MN20A10 were related to the timing of disulfide reduction and the conjugation of the toxin. Most protocols for ADC conjugation require repeated disulfide reductions followed by testing for the number of free thiols. If this is a lengthy process, the free thiols may reoxidize, which may either recreate the original structure or generate new structures resulting in loss of antibody targeting specificity. This situation arose when attempting to conjugate a toxin to the antibody MN20A10, which is an IgG2b isotype. This problem was overcome by 1) empirically determining the molar equivalent required to reduce MN20A10, 2) adding that equivalent amount of reducing agent all at once, and 3) adding the conjugation reagent at an elevated temperature, where the reaction time was further accelerated by reducing the time to reach the desired temperature by performing the reaction in a water bath and by shaking intermittently during the reaction period. Figures 36A-36D show flow cytometry graphs measuring the ability of MNC2 to recognize breast or lung cancer cells before and after conjugation of MMAE. 37A-37D show flow cytometry graphs measuring the ability of MN20A10 to recognize breast or lung cancer cells before and after binding of MMAE.Compared with MN20A10, MNC2 appears to have a lower ability to recognize cancer cells after MMAE binding. Considering the fact that the drug-antibody ratio (DAR) of MNC2-MMAE was higher than that of 20A10-MMAE in this experiment and that MNC2-MMAE had a higher ability to eradicate the same tumors in animals, it is possible that some toxins conjugated to MNC2 antibodies sterically inhibited the binding of the secondary antibody.

[0196] One of the important goals that researchers are trying to achieve is the development of anti-cancer drugs that can recognize and kill early cancer cells that express low levels of the target antigen. Statistically, cancer patients have a higher chance of survival if the cancer is treated at a very early stage. To date, there are few, if any, targeted cancer drugs that can kill early cancer cells with low antigen expression. Modern targeted therapies can only kill cancer cells if the expression of the target antigen exceeds a certain threshold. For example, the anti-breast cancer drug Herceptin™ can only be prescribed to patients with relatively high expression of its target, HER2. Nevertheless, about 20% of patients treated with Herceptin™ develop resistance to Herceptin™ and often also to other anti-cancer drugs. It is now understood that tumor recurrence is frequently caused by cells with low antigen expression that are not killed by the therapeutic agent. When the target antigen is a growth factor receptor, late-stage tumors express high levels of the growth factor receptor, which causes the tumor to grow at a faster rate than cells that express low levels of the growth factor receptor.

[0197] Therefore, although rarely done, it is crucial to test anti-cancer drugs against both early stage cancer cells with low antigen expression and late stage cancer cells expressing high levels of the target antigen. In Figures 38A-45D, the ability of MNC2-ADC and MMN20A10-ADC to kill multiple cancer subtypes was tested, and the tumors were either low-medium antigen expressing cancer cells or high antigen expressing cancer cells. Surprisingly, MNC2-ADC and MN20A10-ADC showed remarkable ability to kill both low and high antigen expressing cancer cells. Low expressing cancer cells were killed by treatment with either MNC2-ADC or MN20A10-ADC in the high nanomolar range of ADCs, which are considered druggable. Cancer cells that expressed high levels of MUC1* were killed with IC50 in the single digit nanomolar range. Sub-micromolar levels of antibodies are considered druggable. Again, this dose range would be very well tolerated due to the high degree of cancer specificity of MNC2 and MN20A10. In vitro measurements of ADC killing often underestimate the potential for ADC killing in vivo. In vivo, killing due to the "bystander" effect is commonly observed, i.e., when killing cells within the tumor trigger host release of cytokines, macrophages, etc. that greatly increase killing. Such effects do not occur in vitro.

[0198] For example, MNC2-MMAE, MNC2-MMAF, MN20A10-MMAE, and MN20A10-MMAF were tested in vitro for their ability to kill T47D wild-type breast cancer cells expressing low levels of MUC1*, and T47D-MUC1* cells engineered to express more MUC1*. Similarly, anti-MUC1*-ADCs were tested for their ability to kill HPAF II wild-type pancreatic cancer cells expressing low levels of MUC1*, and HPAF II-MUC1* cells engineered to express more MUC1* (Figures 38A-38F). In these experiments, the DAR of MNC2-MMAE is comparable to that of MNC2-MMAF, 4.1 vs. 3.7. There is a slight difference between the DAR of MN20A10-MMAE, 5.8, and that of MN20A10-MMAF, 3.8, which is reflected in the killing potency of both. As can be seen, both wild type, low antigen expressing cells are killed when treated with ADC at submicromolar doses, which is considered druggable. Surprisingly, even though MUC1* is a growth factor receptor, the more MUC1* expressed, the greater the ADC-mediated killing effect. Both breast and pancreatic cancer cells engineered to express more MUC1* were readily killed by very low nanomolar concentrations of MNC2-MMAE, MNC2-MMAF, MN20A10-MMAE, and MN20A10-MMAF. Target cell killing by MNC2-deruxtecan or MN20A10-deruxtecan was compared to MNC2-MMAE, MNC2-MMAF, MN20A10-MMAE, and MN20A10-MMAF (Figures 40A-40C). All five anti-MUC1* ADCs appear to have comparable killing potency in this in vitro assay, although bystander cell killing cannot be determined by this assay. Anti-MUC1*-ADCs were also shown to potently kill MUC1*-positive prostate cancer cells (Figures 41A-41B) and non-small cell lung cancer cells (Figures 42A-42B).

[0199] In a set of flow cytometry experiments, the specific killing efficacy of MNC2-MMAE or MN20A10-MMAE is determined by measuring the viability of target cancer cells after addition of MUC1*-ADC. Here, the target cells are T47D wild-type breast cancer cells or T47D breast cancer cells engineered to express more MUC1*, referred to as T47D-MUC1*. As seen in Figure 43, both MN20A10-MMAE and MNC2-MMAE effectively kill both wild-type breast cancer cells and cells engineered to express more MUC1*. However, for both MUC1*-ADCs, the killing efficacy is greater when the cells overexpress the target antigen. Cancer cells expressing low levels of MUC1* are consistent with early stage cancer, whereas cells expressing high levels of MUC1* are consistent with late stage cancer.

[0200] To demonstrate that any cell expressing MUC1* is killed by the MUC1*-ADC described herein, we engineered the MUC1-negative colon cancer cell line HCT-116 to express MUC1*, e.g., HCT-MUC1*. What is seen in the cell viability assay in Figure 44 is that only MUC1*-expressing cells are killed. After adding either unconjugated MN20A10 or MN20A10-MMAE, the viability of MUC1*-expressing cancer cells was measured. As seen in Figure 45, MN20A10-MMAE effectively killed non-small cell lung cancer cells, DU145 hormone-resistant prostate cancer cells, and pancreatic cancer cells.

[0201] Figures 46-48 show magnified images of various types of cancer cells after treatment with either MNC2-ADC or MN20A10-ADC. The killing effect can be easily recognized as a significant reduction in cell number, a change in cell morphology from a characteristic flattened to a small rounded morphology, and floating of cells. In contrast, control wells showed a confluent monolayer of compact cells with a normal flattened morphology, and no floating dead cells were observed. Figures 46A-46C and Figures 46G-46F show T47D-MUC1* breast cancer cells treated with MNC2-MMAE. Figures 46D-46F and Figures 46G-46J show T47D-MUC1* breast cancer cells treated with MNC2-deruxtecan. Figures 47A-47B show DU145 hormone-resistant prostate cancer cells treated with MNC2-MMAE. Figures 47C-47D show DU145 hormone-resistant prostate cancer cells treated with MNC2-deruxtecan. Figures 48A-48B and Figures 48G-48H show T47D-MUC1* breast cancer cells treated with MN20A10-MMAE. Figures 48C-48D and Figures 48I-48J show T47D-MUC1* breast cancer cells treated with MN20A10-deruxtecan. Figures 48K-48L show DU145 hormone-resistant prostate cancer cells treated with MN20A10-MMAE. Figures 48M-48N show DU145 hormone-resistant prostate cancer cells treated with MN20A10-deruxtecan. As is visually evident, the killing effect of anti-MUC1*-ADC shown in Figures 46-48 is consistent with the killing measured by flow cytometry shown in Figures 38-45.

[0202] MNC2-ADC and MN20A10-ADC were assayed for their ability to kill both low and high MUC1* expressing cells by monitoring killing in real time with the xCELLigence instrument (Figures 53-56). In the xCelligence system, adherent target cancer cells are plated on an electrode array 96-well plate. The adherent cells insulate the electrodes and increase the impedance. The number of attached cancer cells is directly proportional to the impedance. Antibodies and antibody drug conjugates are much smaller and do not contribute significantly to the impedance. Thus, an increase in impedance reflects cancer cell proliferation and a decrease in impedance reflects cancer cell killing.

[0203] The breast cancer cell line T47D-wt expresses low to low-moderate levels of MUC1*. The pancreatic cancer cell line HPAF II-wt expresses even lower levels of MUC1*, and the lung cancer cell line NCI-H1975 expresses even lower levels of MUC1*. MNC2 and MN20A10 conjugated to MMAE, MMAF, deruxtecan, or exatecan both effectively killed low-expressing T47D-wt breast cancer cells (Figures 49 and 54), non-small cell lung cancer cells (Figures 51 and 56), and pancreatic cancer cells (Figure 52). Cancer cells expressing low levels of MUC1*, consistent with early stage cancer cells, are effectively killed by MNC2-ADC and MN20A10-ADC when administered at mid-nanomolar doses. As can be clearly seen in Figures 54 and 56, when MNC2-exatecan (DAR 8.2) is compared to MNC2-deruxtecan (DAR 4.0) and MNC2-MMAE (DAR 4.1), MNC2-exatecan exhibited more potent killing of low MUC1* expressing cancer cells, likely due to the presence of more toxin attached.

[0204] In the next set of experiments, MNC2-ADC and MN20A10-ADC are tested for their ability to kill cancer cells expressing high levels of MUC1*. Since MUC1* is the growth factor receptor that drives the proliferation of these cells, it is expected that the higher the level of MUC1*, the more difficult it will be to kill the cells. Unexpectedly, experiments show that the more MUC1* is expressed, the easier it is to kill the cells. We also compared MNC2 and MN20A10 conjugated to MMAE, MMAF, deruxtecan, or exatecan for killing cancer cells expressing high levels of MUC1* using the xCELLigence real-time killing assay. The breast cancer cell lines T47D-MUC1* and HPAF II-MUC1* have been engineered to express high levels of MUC1* consistent with late-stage cancer. As shown in Figures 50 and 53, and Figure 55, cancer cells expressing high levels of MUC1* are completely killed at very low nanomolar doses of 6 nM to 19 nM.S.

[0205] In addition to the in vitro assays, in vivo experiments were also performed in which anti-MUC1* antibodies MNC2 and MN20A10 conjugated with MMAE, MMAF, deruxtecan, or exatecan were administered to test mice implanted with breast, lung, or pancreatic tumors with high or low MUC1* expression. These experiments are more fully detailed in experiment 18. In one example, NOD / SCID / GAMMA mice were implanted with T47D breast cancer cells, either wild type or engineered to express more MUC1*. MNC2-MMAE, DAR3.5, was injected into the animals three times at an initial dose of 5 mg / kg, then increased to 10 mg / kg. As seen in Figure 57, mice with tumors expressing high levels of MUC1*, consistent with late-stage cancer, were effectively eliminated by day 26. Mice implanted with tumors with low MUC1* expressing cells, consistent with early-stage cancer, were not completely eliminated, but the tumor volume was small compared to the control group. In comparison (Figure 58), animals given the same dose of MN20A10-MMAE, DAR2.96 did not show complete tumor disappearance, likely due to the smaller number of toxins attached. The same dose of either MNC2-MMAE (Figure 59) or MN20A10-MMAE (Figure 60) was given to animals implanted with non-small cell lung cancer tumors. MNC2-MMAE, DAR4.1 was highly effective in tumor disappearance in test mice implanted with pancreatic tumors that were either high or low MUC1* expressers (Figure 61). Figure 62 shows an updated version of the experiment shown in Figure 61. After 66 days, animals treated with MNC2-MMAE were tumor-free from day 18 onwards, with no tumor recurrence. MNC2-deruxtecan was given to mice implanted with human breast cancer cells expressing low or high levels of MUC1* (Figures 63 and 64). High MUC1* expressing tumors were essentially gone by day 26. Mice implanted with low MUC1* expressing cells showed a significant reduction compared to controls, but may require higher doses or repeated injections.

[0206] In animal models, we have shown that as tumors progress to later stages, their expression of MUC1* increases. Figures 65A-65D show images of tumor cells. Staining of day 0 cells shows that the cells express more full-length MUC1 than MUC1*, with weaker staining intensity and fewer MUC1* receptors, indicative of early cancer. It can be easily recognized that 62 days after tumor implantation, which corresponds to about 7 years in human time, the expression of MUC1* has changed significantly in late tumors from low to high expression in terms of the degree of expression and the intensity of expression. In contrast, staining of serial sections of tumors shows that full-length MUC1 is expressed, which is expected because it is cleaved to MUC1* after surface expression. However, the staining intensity does not increase, indicating that the majority of the expressed MUC1 has been cleaved to the growth factor receptor type MUC1* in late tumors.

[0207] antibody In this context, an antibody (Ab) that binds to a polypeptide of interest as a "binding" is understood by those skilled in the art. For example, an antibody, or a conjugate comprising such an Ab, as described herein, may generally bind to other polypeptides or proteins with low affinity, as measured, for example, by immunoassays or other assays known in the art. In certain embodiments, an Ab, or a conjugate comprising such an Ab that specifically binds to a polypeptide of interest, as described herein, binds to the polypeptide of interest with an affinity that is at least 2 log, 2.5 log, 3 log, 4 log, or more than the affinity with which the Ab or conjugate binds to another polypeptide. In another particular embodiment, an Ab, or a conjugate comprising such an Ab, as described herein, does not specifically bind to a polypeptide other than the polypeptide of interest. In certain embodiments, an Ab, or a conjugate comprising such an Ab, as described herein, specifically binds to a polypeptide of interest with an affinity (Kd) of 20 mM or less. In certain embodiments, such binding has an affinity (Kd) of about 20 mM or less, about 10 mM or less, about 1 mM or less, about 100 μM or less, about 10 μM or less, about 1 μM or less, about 100 nM or less, about 10 nM or less, or about 1 nM or less. Unless otherwise specified, "binds", "binds to", "specifically binds", or "specifically binds to" in this context are used interchangeably.

[0208] In some embodiments, the target cell is a cancer cell, hi some embodiments, the cancer is breast cancer, colon cancer, prostate cancer, pancreatic cancer, or lung cancer.

[0209] In some embodiments, the antibody binds to cancer cells that express MUC1* on their surface.

[0210] In certain embodiments, the antibody comprises about 10, about 20, about 30, about 40, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, or about 950 amino acids.

[0211] In certain embodiments, the antibody comprises about 10-50, about 50-100, about 100-150, about 150-200, about 200-250, about 250-300, about 300-350, about 350-400, about 400-450, about 450-500, about 500-600, about 600-700, about 700-800, about 800-900, or about 900-1000 amino acids.

[0212] In some embodiments, the conjugate comprises an antibody Ab. In some embodiments, the Ab is a monoclonal antibody. In some embodiments, the Ab is a human antibody. In some embodiments, the Ab is a humanized antibody. In some embodiments, the Ab is a chimeric antibody. In some embodiments, the Ab is a full-length antibody comprising two heavy chains and two light chains. In certain embodiments, the Ab is an IgG antibody, e.g., an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the Ab is a single-chain antibody. In yet other embodiments, the Ab is an antigen-binding fragment of an antibody, e.g., a Fab fragment.

[0213] In certain embodiments, the Ab is an IgG1 antibody. In certain embodiments, the Ab is an IgG2b antibody.

[0214] In some embodiments, the antibody specifically binds to a cell surface protein. In some embodiments, the antibody specifically binds to a cell surface receptor. In some embodiments, the antibody specifically binds to a cell surface receptor ligand.

[0215] In some embodiments, the antibody, or functional fragment or variant thereof, specifically binds to MUC1*, hi some embodiments, the antibody comprises an anti-MUC1* heavy chain and an anti-MUC1* light chain.

[0216] In some embodiments, the anti-MUC1* heavy chain comprises an anti-MUC1* heavy chain variable domain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, the anti-MUC1* light chain comprises an anti-MUC1* light chain variable domain. In some embodiments, the anti-MUC1* light chain variable domain comprises the variable domain of a kappa or lambda light chain.

[0217] In some embodiments, the anti-MUC1* heavy chain variable domain comprises a variable domain of an IgG1 heavy chain and the anti-MUC1* light chain variable domain comprises a variable domain of a kappa or lambda light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises a variable domain of an IgG2 heavy chain and the anti-MUC1* light chain variable domain comprises a variable domain of a kappa or lambda light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises a variable domain of an IgG3 heavy chain and the anti-MUC1* light chain variable domain comprises a variable domain of a kappa or lambda light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises a variable domain of an IgG4 heavy chain and the anti-MUC1* light chain variable domain comprises a variable domain of a kappa or lambda light chain.

[0218] In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG1 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a kappa light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG2 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a kappa light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG3 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a kappa light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG4 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a kappa light chain.

[0219] In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG1 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a lambda light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG2 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a lambda light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG3 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a lambda light chain. In some embodiments, the anti-MUC1* heavy chain variable domain comprises the variable domain of an IgG4 heavy chain and the anti-MUC1* light chain variable domain comprises the variable domain of a lambda light chain.

[0220] In some embodiments, an antibody, or functional fragment or variant thereof, that specifically binds to MUC1* comprises a single chain variable fragment (scFv) or an antigen-binding fragment (Fab). In some embodiments, an antibody, or functional fragment or variant thereof, that specifically binds to MUC1* comprises a single chain variable fragment. In some embodiments, an antibody, or functional fragment or variant thereof, that specifically binds to MUC1* comprises an antigen-binding fragment (Fab).

[0221] In some embodiments, the anti-MUC1* heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the anti-MUC1* heavy chain variable domain comprise the amino acid sequences set forth in HC-CDR1: SEQ ID NO: 1 or 4, HC-CDR2: SEQ ID NO: 2 or 5, and HC-CDR3: SEQ ID NO: 3 or 6, and the CDRs comprise 0 to 2 amino acid modifications (e.g., 0 or 1 amino acid modifications) in at least one of HC-CDR1, HC-CDR2, or HC-CDR3.

[0222] In some embodiments, the anti-MUC1* light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the anti-MUC1* light chain variable domain comprise the amino acid sequences set forth in LC-CDR1: SEQ ID NO: 13 or 16, LC-CDR2: SEQ ID NO: 14 or 17, and LC-CDR3: SEQ ID NO: 15 or 18, and the CDRs comprise 0 to 2 amino acid modifications (e.g., 0 or 1 amino acid modifications) in at least one of LC-CDR1, LC-CDR2, or LC-CDR3.

[0223] In some embodiments, the anti-MUC1* heavy chain comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 38 or 44. In some embodiments, the anti-MUC1* light chain comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 41 or 47.

[0224] Antibody drug conjugates (ADCs) work when the variable region of the antibody portion recognizes a molecule expressed on the outside of a cancer cell. After the antibody binds to the target antigen, the entire ADC is internalized. Antibody internalization is a prerequisite for the ADC's cell killing function. In many cases, cellular internalization of the ADC biochemically alters the toxin, making it more potent or the changes may trap the toxin inside the cell and reduce off-target toxicity. Not all receptors are internalized after ligand binding, or especially after antibody binding, which may dimerize the receptor, making internalization more difficult, if not impossible. Figures 25A-25D are photographs taken with a confocal microscope demonstrating that an anti-MUC1* antibody, such as MNC2, is internalized after binding to the extracellular domain of the MUC1* receptor.

[0225] payload Antibody drug conjugates (ADCs) combine the targeting specificity of an antibody (e.g., a monoclonal antibody) with the potency of a small molecule drug (known as the payload or cytotoxic group) by linking them together in a single ADC molecule that retains the properties of both. The improved selectivity and potency of ADCs provide superior safety and efficacy compared to traditional chemotherapeutic drugs, resulting in a broader therapeutic window. The terms "payload" and "cytotoxic group" are used interchangeably herein.

[0226] In some embodiments, the payload is a topoisomerase inhibitor. In some embodiments, the topoisomerase inhibitor is a topoisomerase I inhibitor. In some embodiments, the topoisomerase inhibitor is exatecan or a derivative thereof. In some embodiments, the topoisomerase inhibitor is deruxtecan or a derivative thereof.

[0227] In some embodiments, the payload is a tubulin formation inhibitor. In some embodiments, the tubulin formation inhibitor is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0228] Some of the more recent toxic payloads used in ADC formats, such as deruxtecan, belong to the exatecan family of topoisomerase I inhibitors. One such recent payload binder configuration used in ADC formats is deruxtecan, shown in Figure 28, where a maleimidocaproyl (MC) moiety facilitates attachment to a cysteine ​​on the antibody. Maleimidocaproyl is attached to the toxic payload Dxd via a binder HN-CH2- that binds to a glycine phenylalanine linker GGFG, Dxd. Dxd is a topoisomerase I inhibitor, a mechanism that inhibits cell division. In another example shown in Figure 29, the exatecan payload is attached to the antibody via a para-aminobenzyl (PAB) moiety, which is attached to a valine-citrulline (VC) moiety, which in turn is attached to a maleimidocaproyl (MC) moiety that facilitates attachment to a cysteine ​​on the antibody.

[0229] Exatecan has a high level of bystander effect, meaning that it can kill neighboring cells. Clinical trials of Enhertu, a novel ADC targeting HER2+ breast cancer, were plagued by severe and life-threatening side effects, including pneumonia. It has been reported that up to 16% of Enhertu patients suffered from treatment-induced pneumonia. However, Enhertu received FDA approval because it reportedly extended the survival of metastatic breast cancer patients by 2 years. It is not entirely clear whether these side effects are due to the payload or to the target, HER2, which is also expressed in normal lungs and normal hearts. Recall that the first two patients treated with a HER2-targeted CAR T cell product died shortly after infusion. Subsequent studies showed that the lethality was due to the use of an antibody with extremely high affinity for HER2. Relatively recently, yet another patient died after being treated with a different HER2-targeted ADC. These results highlight the importance of selecting antibodies with superior cancer selectivity to avoid such life-threatening toxicities. MNC2 and MN20A10 unexpectedly possessed a very high degree of cancer specificity and did not induce toxicity in animal studies, where each antibody was incorporated into several ADC formats.

[0230] Linker In certain embodiments, the linker-L- comprises one or more of a carbon atom, a nitrogen atom, a sulfur atom, an oxygen atom, and combinations thereof. In certain embodiments, the linker-L- comprises one or more amino acids. In certain embodiments, the linker-L- comprises one or more of an ether bond, a thioether bond, an amine bond, an amide bond, a carbon-carbon bond, a carbon-nitrogen bond, a carbon-oxygen bond, a carbon-sulfur bond, and combinations thereof. In certain embodiments, the linker-L- comprises a linear structure. In certain embodiments, the linker-L- comprises a di-peptide, a tri-peptide, or a tetra-peptide peptide bond moiety.

[0231] The linker may include at least one glycine. The linker may include at least one glycine and a phenylalanine. The linker may include

[0232] [ka] The linker may include a structure of: The linker may include valine. The linker may include citrulline. The linker may include valine and citrulline. The linker may include a structure of:

[0233] [ka] The dipeptide linking moiety may have the structure:

[0234] In certain embodiments, Z is a conjugate moiety capable of forming a covalent bond with an amino acid of a polypeptide. Z can be attached to the N-terminus of the linker. Z can comprise a maleimide. In certain embodiments, the amino acid is cysteine. In certain embodiments, Z is

[0235] [ka] In one embodiment, Z is

[0236] [ka] In one embodiment, Z is

[0237] [ka] It is.

[0238] The linker may comprise a linking moiety, R. In certain embodiments, R is a linking moiety that can attach a payload to the C-terminus of the linker. R is

[0239] [ka] where * indicates the point of attachment of the payload. R can include a moiety having the structure:

[0240] [ka] where * indicates the point of attachment of the payload. R can include a group of the structure:

[0241] [ka] where * indicates the point of attachment of the payload (e.g., a cytotoxic group). R can include a group having the structure:

[0242] [ka] where * indicates the point of attachment of the payload.

[0243] Figure 26 shows the chemical structure of the payload monomethyl auristatin E, i.e. MMAE, as well as useful linker and reactive molecules that facilitate chemical conjugation of the payload to an antibody. In this example, MMAE is attached to the antibody via a para-aminobenzyl (PAB) moiety, which is attached to a valine-citrulline (VC) moiety, which in turn is attached to a maleimidocaproyl (MC) moiety, which facilitates conjugation with a cysteine ​​on the antibody. When the ADC is internalized, cathepsin B enzymatically cleaves the payload from the antibody, which is facilitated by both PAB and VC. The payload, monomethyl auristatin E (MMAE), inhibits cell division by blocking the polymerization of tubulin.

[0244] Monomethylauristatin F, referred to as MMAF, is another example of a toxic payload that inhibits cell division by blocking the polymerization of tubulin. In the example shown in FIG. 27, the payload, MMAF, is attached to the antibody via a para-aminobenzyl (PAB) moiety, which is attached to a valine-citrulline (VC) moiety, which in turn is attached to a maleimidocaproyl (MC) moiety, which facilitates conjugation with a cysteine ​​on the antibody. Once the ADC is internalized, cathepsin B enzymatically cleaves the payload from the antibody. Unlike MMAE, MMAF contains a carboxylic acid, which makes it difficult for the payload to exit the cell after it is cleaved from the antibody.

[0245] Some of the more recent toxic payloads used in ADC formats, such as deruxtecan, belong to the exatecan family of topoisomerase I inhibitors. One such recent payload binder configuration used in ADC formats is deruxtecan, shown in Figures 28A-28E, where a maleimidocaproyl (MC) moiety facilitates attachment to a cysteine ​​on the antibody. The maleimidocaproyl is attached to the toxic payload, Dxd, via a binder HN-CH2- that binds to a glycine phenylalanine linker, GGFG, Dxd. Dxd is a topoisomerase I inhibitor, a mechanism that inhibits cell division. In another example, shown in Figures 29A-29E, the exatecan payload is attached to the antibody via a para-aminobenzyl (PAB) moiety, which is attached to a valine-citroline (VC) moiety, which in turn is attached to a maleimidocaproyl (MC) moiety that facilitates attachment to a cysteine ​​on the antibody.

[0246] Pharmaceutical Compositions In another aspect, provided herein is a pharmaceutical composition comprising a conjugate (e.g., ADC) and a multispecific antibody as disclosed herein. In some embodiments, the pharmaceutical composition comprises a conjugate of formula (I) and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a multispecific antibody as disclosed herein and a pharma- ceutically acceptable carrier.

[0247] The pharmaceutical composition herein is formulated with one or more physiologically acceptable carriers, including excipients and auxiliary agents, which are used pharmacy and facilitate the processing of active agent into preparations.Suitable formulations vary according to the route of administration selected.

[0248] In certain embodiments, the pharmaceutical compositions disclosed herein further comprise a pharma- ceutically acceptable diluent, excipient, or carrier. In some embodiments, the pharmaceutical compositions comprise other medicinal or pharmaceutical agents, carriers, adjuvants such as preservatives, stabilizers, wetting agents, or emulsifiers, solution promoters, salts for adjusting osmotic pressure, and / or buffers.

[0249] In certain embodiments, the pharmaceutical compositions disclosed herein are administered to a subject by any suitable route of administration, including but not limited to parenteral (intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravitreal, infusion, or topical) administration.

[0250] Preparations suitable for intramuscular, subcutaneous, peritumoral or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, cremophor, etc.), suitable mixtures thereof, vegetable oils (olive oil, etc.), and injectable organic esters such as ethyl oleate. Proper fluidity is maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Preparations suitable for subcutaneous injection also include any additives such as preservatives, wetting agents, emulsifying agents, and dispensing agents.

[0251] For intravenous injections, the active agents are optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.

[0252] Parenteral injections optionally include bolus injection or continuous infusion. Preparations for injection are optionally provided in unit dosage form, for example, in ampoules or multi-dose containers, with additional preservatives. In some embodiments, the pharmaceutical compositions described herein are in a form suitable for parenteral injection, such as a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and contain formulatory agents, such as suspending agents, stabilizing agents and / or dispersing agents. Pharmaceutical preparations for parenteral administration include aqueous solutions of the active agent in water-soluble form. In addition, suspensions are optionally prepared as suitable oily injection suspensions.

[0253] In some embodiments, the pharmaceutical compositions described herein are in unit dosage form suitable for single administration of precise dosage amounts. In unit dosage form, the formulation is divided into unit doses containing the appropriate amount of active agent disclosed herein. In some embodiments, the unit dose is a packaged form containing a discrete amount of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. In some embodiments, aqueous suspension compositions are packaged in single-dose non-reclosable containers. Alternatively, multi-dose reclosable containers are used, in which case it is common to include a preservative in the composition. By way of example only, formulations for parenteral injection are presented in unit dosage form, including but not limited to ampoules, or in multi-dose containers with additional preservatives. EXAMPLES

[0254] Example 1. Anti-MUC1* internalization experiments Internalization of MNC2 was demonstrated (Figures 25A-25B).

[0255] 8-well chamber slides (Nunc™ Lab-Tek™ II Chamber Slide™ System Thermo Cat. No. 154534PK or Ibidi™ 8-well μ-Slide ibiTreat: #1.5 polymeric coverslip, tissue culture treated, sterile) were coated with collagen (Sigma #C3867) where 300 μL of collagen was added per well, incubated overnight at 4° C., washed once with PBS, then once with water, then air-dried in a biosafety hood. Allow wells to dry under the hood.

[0256] T47D breast cancer cells were plated at 30,000–50,000 cells / well in 10% RPMI and cultured for 48 h or until approximately 70% confluent. Media was removed and cells were serum starved in 2% RPMI for 24 h. Cells were washed with ice-cold PBS.

[0257] Antibodies were diluted in cold PBS+2%FBS to a final concentration of 300ug / mL. 200ul of antibody solution was added to each well and incubated for 2 hours at 4°C in the dark on a rocking platform shaker at speed 6. Cells were then washed 3 times with cold PBS.

[0258] Anti-mouse Alexa488 or anti-human Alexa555 antibodies at 10ug / mL were diluted 1:200 in PBS containing 2% FBS and incubated with shaking in the dark for 1 hour at 4°C. Cells were washed 3 times with cold PBS.

[0259] For internalization, warm 10% RPMI + 75 nM lysotracker deep red (1 / 13,333) was added and incubated for 2 hours at 37°C in a CO2 incubator. Cells were washed 3 times with cold PBS.

[0260] Cells were fixed with fresh 4% formaldehyde in PBS for 15 min at room temperature, then washed 3 times with cold PBS. Hoescht dye 33342 in PBS was added at 1ug / mL and incubated for 10 min at room temperature, after which the medium was removed without a washing step. For Nunc™ Lab-Tek™, cover slips (#1.5) were mounted in chamber slides with mounting medium (Prolong Diamond antifade), avoiding air bubbles. Slides were then stored for 24 hours at room temperature on a flat surface. Long-term storage was done in the dark at 4°C. For Ibidi slides, Ibidi mounting medium is added.

[0261] Photographs were taken with a confocal microscope.

[0262] Example 2. MNC2-ADC or MN20A10-ADC (Overview of Small Scale (3 mg) Preparation of Antibody-Drug Conjugates) Solutions of unconjugated MNC2, an IgG1 antibody, or MN20A10, an IgG2b antibody, were reduced with excess dithiothreitol (DTT) in borate buffer at pH 8.0. After reduction for 2 hours at 37°C, aliquots were tested for the amount of free thiols present using Ellman's thiol test. Depending on the antibody isotype, the number of free thiols varies. If analysis of free thiols using Ellman's reagent showed an insufficient number of free thiols, more DTT was added and incubated for an additional period at 37°C. The process of measuring free thiols and adding more reducing agent was repeated until a sufficient number of free thiols was obtained. MN20A10 required more reducing agent and a longer reduction time than MNC2. The reduced antibody was then placed in 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 6.0 and the free cysteines were alkylated with 11 molar equivalents of maleimide-conjugated toxin for approximately 1 hour. The crude ADC was then purified by a desalting column to remove excess toxin, low molecular weight contaminants such as DMSO and other small molecular weight contaminants. The drug-antibody ratio (DAR) was calculated from the ratio of the UV absorbance of the toxin at 248 nm (or 370 nm for deruxtecan) to the antibody absorbance at 280 nm. Analysis of the conjugates to determine the drug-antibody ratio (DAR), i.e., the [drug:mAb] molar ratio, was performed by hydrophobic interaction chromatography-high performance liquid chromatography according to published methods (Effects of drug loading on antitumor activity of a monoclonal antibody drug conjugate. Hamblett, KJ, Senter, PD, Chace, DF, Sun, MMC, Lenox, J., Cerveny, CG, Kissler, KM, Bernhardt, SX, Kopcha, AK, Zabinski, RF, Meyer, DL, and Francisco, JAClin Cancer Res. 2004 Oct 15;10(20):7063-70. DOI:10.1158 / 1078-0432.CCR-04-0789).

[0263] Following this procedure, small scale preparations of MNC2-MMAE, MNC2-MMAF, MNC2-deruxtecan, MNC2-exatecan, MN20A10-MMAE, MN20A10-MMAF, MN20A10-deruxtecan and MN20A10-exatecan were generated.

[0264] Example 3: Detailed example of the production of a small scale (3 mg) preparation of MNC2-deruxtecan IgG1 MNC2 antibody (3.00 mg, 1.578 mL, 1.9 mg / mL, 20 nM) was thawed from -80° C. for 1 minute in a 37° C. water bath. For mouse IgG1 antibody, protein concentration was determined by Nanodrop using an extinction coefficient of 14,000 L / g-cm. The antibody solution was concentrated using a centrifugal filter (Amicon Ultra 0.5ML 50,000mw cut off (cat. no. UFC505024) (14,000xg, 4°C for 8 minutes) to obtain a volume of 100μL or less. The buffer was then exchanged into pH 8.0 borate buffer (25mM sodium tetraborate, 25mM NaCl, 1mM EDTA) by concentrating to less than 100μL and resuspending in approximately 300μL of fresh buffer. This process was repeated a total of 3 times. The concentrated antibody (approximately 100ul) was transferred to a 0.5mL screw cap centrifuge tube and made up to 300μL with pH 8.0 borate buffer (25mM sodium tetraborate, 25mM sodium chloride, 1mM EDTA) to make a 10mg / mL antibody solution.

[0265] A solution of dithiothreitol (DTT) (6.48 mM) was prepared by weighing out DTT (5 mg, 32.4 mmol) and dissolving it in 5 mL, pH 8.0 borate buffer (25 mM sodium tetraborate, 25 mM sodium chloride, 1 mM EDTA). The DTT solution (6.48 mM, 30.9 uL, 200 nmol, 10 equiv.) was added to the antibody solution and the tube was heated in a 37°C water bath for 2 hours. The warm reaction mixture was vortexed periodically every 15 minutes. After 2 hours, an aliquot (6 μL) of the reaction mixture was removed for Ellman's thiol analysis, which revealed 7.68 free thiols per antibody out of a total of 8 thiols that could be present for IgG1.

[0266] The antibody reduction mixture was cooled to 4°C in an ice bath, the solution was transferred to a centrifugal filter (Amicon Ultra 0.5ML 50,000mw cutoff) and the buffer was switched to MES buffer (50mM) pH 6.0 three times [14,000xg, 4°C for 8 min]. The concentrated reduced antibody was diluted to 640μL with MES buffer (50mM) pH 6.0.

[0267] A stock solution of deruxtecan in DMSO (10 mg / mL, 9.7 mM) was prepared by weighing out 1.80 mg, 1.74 μmol and dissolving it in DMSO (180 uL). A solution of deruxtecan in 50% DMSO / MES pH 6.0 buffer (50 mM) (2 mM, 110.2 uL, 220 nmol) was prepared by diluting deruxtecan solution (22.8 uL, 10 mg / mL in DMSO) with DMSO (32.3 uL) and then further diluting with 50 mM MES pH 6.0 (50 mM, 55.1 uL) to make a solution of 2 nM deruxtecan in 50% DMSO / 50% MES pH 6.0 buffer (50 mM). The deruxtecan solution (110.1 uL, 50% DMSO / 50% MES buffer pH 6.0, 2 mM, 73.4 nmol, 11 equiv.) was added to the tube containing 640 μL of reduced antibody. The reaction was mixed slowly at room temperature for 1 hour on a vertical rotor and monitored by hydrophobic interaction chromatography (HIC) HPLC, which indicated whether unreacted antibody remained. The retention time of traces of unreacted antibody was compared to the reaction product to determine whether unreacted antibody remained, and if so, the reaction was allowed to continue. For small scale, the time is typically 1-2 hours. Large scale preparations may require reaction for 8-12 hours.

[0268] After approximately 1 hour, the reaction was purified on a desalting column (Cytiva PD miditrap g-25 medium, catalog number 28918008). The top cap of the column was removed and the loading buffer was discarded. The bottom cap of the column was removed and the column was conditioned with Gibco PBS pH 7.4 (ref. 10010-023) (3x5mL) by gravity flow-through. The antibody solution (750uL) was loaded onto the column, allowing it to completely enter the resin bed, followed by PBS (250uL). The purified ADC was then eluted from the column with PBS pH 7.4 (1.25mL). Protein concentration measurement by nanodrop indicated 1.76mg / mL antibody in 1.25mL PBS, i.e. 2.2mg (73% yield) of conjugated antibody was recovered from the initial 3.0mg of unconjugated antibody. The purified ADC was analyzed by UV-VIS spectroscopy at 370 nm and 280 nm, giving a drug-to-antibody ratio (DAR) of 6.7.

[0269] Example 4: MN20A10-Deruxtecan MN20A10-deruxtecan was prepared in a similar manner but with 20 equivalents of DTT, which gave 4.77 free thiols per antibody. After conjugation with deruxtecan, the purified ADC showed a DAR of 4.60 by UV-VIS spectroscopy at 370 nm and 280 nm.

[0270] Example 5: MNC2-MC-VC-PAB-MMAE MNC2-MC-VC-PAB-MMAE was prepared in a similar manner but with 5 equivalents of DTT, which gave 6.12 free thiols per antibody. After conjugation with MC-VC-PAB-MMAE, the purified ADC showed a DAR of 4.39 by HIC-HPLC and 5.12 by UV-VIS spectroscopy at 248 nm and 280 nm.

[0271] Example 6: MNC2-MC-VC-PAB-MMAF MNC2-MC-VC-PAB-MMAF was prepared in a similar manner but with 5 equivalents of DTT, which gave 4.53 free thiols per antibody. After conjugation with MC-VC-PAB-MMAF, the purified ADC showed a DAR of 3.65 by HIC-HPLC.

[0272] Example 7: MNC2-MC-VC-PAB-Exatecan MNC2-MC-VC-PAB-exatecan was prepared in a similar manner but with 10 equivalents of DTT, which gave 6.20 free thiols per antibody. After conjugation with MC-VC-PAB-exatecan, the purified ADC showed a DAR of 8.40 by UV-VIS spectroscopy at 370 nm and 280 nm.

[0273] Example 8: MN20A10-VC-PAB-MMAE MN20A10-MC-VC-PAB-MMAE was prepared in a similar manner but with 7.5 equivalents of DTT, which gave 4.08 free thiols per antibody. After conjugation with MC-VC-PAB-MMAE, the purified ADC showed a DAR of 3.81 by HIC-HPLC and 4.11 by UV-VIS spectroscopy at 248 nm and 280 nm.

[0274] Example 9: MN20A10-MC-VC-PAB-MMAF MN20A10-MC-VC-PAB-MMAF was prepared in a similar manner, but with 15 equivalents of DTT to yield 3.87 free thiols per antibody. After conjugation with MC-VC-PAB-MMAF, the purified ADC showed a DAR of 3.79 by HIC-HPLC.

[0275] Example 10. Large scale (30 mg) preparation of MNC2-ADC or MN20A10-ADC antibody-drug conjugates In a similar manner to the small-scale ADC preparation, a solution of unconjugated antibody was reduced with excess dithiothreitol (DTT) in borate buffer at pH 8.0. After 2 hours of reduction, an aliquot was tested for the amount of free thiol present using Ellman's thiol test. The reduced antibody was then placed in MES buffer at pH 6.0 and the free cysteines were alkylated overnight with 11 molar equivalents of maleimide-conjugated poison. The crude ADC was then purified by a desalting column.

[0276] An aliquot of IgG1 MNC2 antibody (15.7 mL of 1.92 mg / mL, 30.1 mg, 200 nmol) was thawed from -80 °C for 1 min in a 37 °C water bath. The concentration was measured by nanodrop using the mouse IgG1 antibody environment with volume integrated and background subtraction turned off. 30 mg of antibody (1.9 mg / mL 200 nmol) in 16.17 mL. The solution was concentrated using three centrifugal filters (Amicon Ultra 4 mL 30,000 mw cutoff (ref. UFC803024)), buffer exchanged three times into borate buffer pH 8.0 (25 mM sodium tetraborate, 25 mM NaCl, 1 mM EDTA) and spun at 4000 g for 15-25 min in an Eppendorf 5804R centrifuge with a swinging rotor. The concentrated antibody was transferred to a 5 mL screw-cap tube and made up to 3 mL with pH 8.0 borate buffer (25 mM sodium tetraborate, 25 mM sodium chloride, 1 mM EDTA) to make a 10 mg / mL antibody solution.

[0277] A solution of DTT (6.48 mM) was prepared by weighing out DTT (5 mg) and dissolving it in pH 8.0 borate buffer (5 mL) (25 mM sodium tetraborate, 25 mM sodium chloride, 1 mM EDTA). The DTT solution (6.48 mM, 617 uL, 4000 nmol, 20 equiv.) was added to the antibody solution and the tube was heated in a shaker incubator at 37°C for 2 hours. After 2 hours, an aliquot (6 uL) of the reaction mixture was removed for Ellman's thiol analysis. Analysis of these results showed 7.12 moles of free thiol per mole of antibody.

[0278] In a similar manner, MN20A10 was reduced with a total of 20 equivalents of DTT for 3 h with several repetitions of the free thiol test.

[0279] The tube of reduced antibody was then cooled to 4 °C in an ice bath and the solution was transferred evenly to three centrifugal filters (Amicon Ultra 4 mL 30,000 ~mw cutoff (ref. UFC803024)), buffer exchanged three times into MES (50 mM) buffer, pH 6.0, and spun at 4000 g in an Eppendorf 5804R centrifuge for 15-25 min in MES (50 mM) pH 6.0. The concentrate was transferred to a 15 mL Falcon tube, the concentrator was rinsed twice with buffer, and the final volume was brought to 6400 µL with MES (50 mM) buffer, pH 6.0.

[0280] A stock solution of deruxtecan in DMSO (10 mg / mL, 9.67 mM) was prepared by weighing out deruxtecan (3.4 mg, 3.29 μmol) and dissolving it in DMSO (340 μL). A 1100 μL solution of 2 mM in 50% DMSO / MES pH 6.0 (50 mM) was prepared by diluting the DMSO stock deruxtecan solution (227.5 μL of 9.67 mM, 2.22 μmol) with DMSO (322.5 μL) and then further diluting with MES pH 6.0 (50 mM, 550 μL) to make 1100 μL of 2.068 mg / mL (2 mM) deruxtecan in 50% DMSO / MES pH 6.0 (50 mM). To the tube containing 6400 μL of reduced antibody, deruxtecan solution (2 mM, 1000 uL, 2200 nmol, 11 eq.) was added. The reaction was mixed for 18 hours at room temperature on a vertical rotator and monitored by HIC HPLC after 1 hour and the next day.

[0281] After 18 hours, the reaction was purified on a desalting column (Cytiva Sephadex G-25 medium, catalog number 17003301). Sephadex g-25 medium grade gel (10.13 g) was swollen in Gibco PBS pH 7.4 (Gibco catalog number 10010-023, 50 mL) in a round bottom flask at room temperature for 3 hours. A 25 g Teledyne Isco Redi-Sep sample load cartridge (Isco catalog number 693873240) was attached to a ring stand and the cartridge was primed by injecting PBS from the bottom. After allowing the resin to swell for 3 hours, the flask was degassed three times with argon / vacuum. The swollen resin was then poured into the cartridge and the PBS was eluted by removing the bottom cap. All of the resin was transferred to the cartridge by washing the round bottom flask with PBS. A waste container was placed below the column. The column was conditioned with 3x50mL of Gibco PBS pH7.4 (ref. 10010-023) by gravity flow-through. The filter was placed on top of the cartridge and the column packing device was pressed down, taking care to only slightly compress the gel. The column height was approximately 74mm. The antibody solution (7500uL) was loaded onto the column, allowing it to completely enter the resin bed. PBS (2500uL) was added to the column. A fresh 50mL Falcon tube was placed below the column and the purified ADC was eluted with PBS pH7.4 (14mL). For column efficiency, 1mL fractions were passed until a total volume of 50mL had passed through the column. The collected solution was analyzed by nanodrop, giving an antibody concentration of 1mg / mL in 17.5mL PBS, i.e. 17.5mg of antibody, a yield of 58% from unconjugated MNC2.

[0282] Example 11: MNC2-MMAE MNC2-MMAE was prepared in a similar manner, but with 10 equivalents of DTT to yield 6.32 free thiols per antibody. After conjugation with MC-VC-PAB-MMAE, the purified ADC showed a DAR of 4.10 by HIC-HPLC and 5.20 by UV-VIS spectroscopy at 248 nm and 280 nm. Example 12: MN20A10-MMAE

[0283] MN20A10-MMAE was prepared in a similar manner, but with 20 equivalents of DTT to yield 3.67 free thiols per antibody. After conjugation with MC-VC-PAB-MMAE, the purified ADC showed a DAR of 2.96 by HIC-HPLC and 3.57 by UV-VIS spectroscopy at 248 nm and 280 nm.

[0284] [Table 8]

[0285] Overall, MN20A10 required unexpectedly higher amounts of reducing agent (DTT) to yield the same number of free thiols compared to the MNC2 antibody.

[0286] MN20A10 required 20 equivalents of DTT to give 3.67 to 4.2 free thiols (Examples 11 and 3).

[0287] Scaling up MN20A10 with 20 equivalents of DTT gave less free thiol (3.67) than the smaller scale (4.2).

[0288] Examples 7 and 8 demonstrate the challenges associated with the reduction of MN20A10.

[0289] In Example 7, 7.5 equivalents of DTT gave 4.08 free thiol, whereas in Example 8, using more (15 equivalents) resulted in equal or less reduction (3.87 free thiol).

[0290] In contrast, MNC2 required only 5 equivalents of DTT to yield 4.5-6.1 free thiols (Examples 5 and 4).

[0291] 20 equivalents of DTT were used for MNC2-deruxtecan (3 mg and 30 mg) to force the reaction to the highest possible DAR (Examples 2 and 9).

[0292] To directly compare the results to Example 2, MNC2-MC-VC-PAB-exatecan (Example 6) was reduced with 10 equivalents of DTT.

[0293] Example 13: Ellman's thiol test to measure the number of free thiols upon ADC reduction Dissolve 31.7 mg of Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid, ThermoScientific catalog no. 22582) and 164 mg of sodium acetate in 40 mL of water to prepare a 40 mL solution of 2 mM Ellman's reagent. Keep Ellman's stock solution at 4 °C.

[0294] Desalting of reduced antibody aliquots: Take two spin columns (Zeba spin desalting columns ref. 89882) and break off the bottoms to loosen the caps. Place these in a spin centrifuge tube. Spin in a microcentrifuge (Spectrafuge 24D) at 1.5xg for 1 minute to remove the buffer. Discard the eluted buffer. Condition the column by adding 400μL of buffer (borate buffer pH 8.0 consisting of 25mM sodium tetraborate, 25mM NaCl, 1mM EDTA), condition for 5 minutes and then centrifuge at 1.5xg for 1 minute. Discard the eluate between spins. Repeat conditioning and centrifugation.

[0295] Prepare Ellman's Reagent: Pipette 850 μL of water to make 1 mL of Ellman's Reagent. Add 100 μL of 1M Tris-HCl pH 8.0 followed by 50 μL of the Ellman's stock prepared above. Add 70 μL to the bottom of two new centrifuge tubes (Protein LoBind Tube, Eppendorf Cat. No. 022431102), label one as sample and the other as control. Once the column conditioning is complete, transfer it to the clean new centrifuge tubes labeled as sample and control.

[0296] Sample and control preparation: The final concentration of thiol after dilution should be in the range of 10-50 μM. Antibody reduction reactions are performed at 10 mg / mL or 66.7 uM. Dilutions are 5-fold dilutions bringing the concentration to 13.3 μM. Based on historical data, a figure of approximately 0.24 mAu corresponds to approximately 4 thiols per antibody when the control is approximately 0.03. If additional equivalents of reducing agent are added, the new concentration should be reflected in the next thiol test.

[0297] Prepare the control solution immediately before performing the test. The control shall have the same concentration of DTT or TCEP as the reaction without the antibody present. Pipette 24 μL of borate buffer (or appropriate buffer) into the two tubes labeled Sample Preparation and Control Preparation. Add 6 μL of reduced antibody solution and 6 μL of control solution to each tube. Slowly load 30 μL from each tube into the respective tube with the desalting column. Dispense the liquid to the center, without touching the sides of the desalting resin. Spin the tubes at 1.5 g for 2 minutes and analyze the flow-through in a 100 μL cuvette in a spectrophotometer at 412 nm.

[0298] The concentration of antibody in the thiol test was calculated by using C1xV1=MNC2xV2, where C1 is the initial concentration (66.7 μM), V1 is the 6 μL taken from the sample, and V2 is the 30 μL diluted. Solving for MNC2 gave the concentration of antibody in the thiol assay of 13.34 pM. The absorbance at 412 nm is used to measure the concentration of free thiol in the sample. First, the absorbance of the control is subtracted from the sample. The subtracted absorbance of the control is then fitted to Beer's law A=ebc, where A is the absorbance, e is the molar extinction coefficient of the antibody, b is the path length of the cuvette, and c is the molar concentration. The molar extinction coefficient of Ellman's reagent is 14,150 L / mol-cm. The path length of the cuvette is 1 cm. Calculate the molar concentration of free thiol in the sample.

[0299] Once the concentration is determined, the dilution factor for the 30 µL to 70 µL of Ellman's reagent passed through the column is 3.333. Calculate the number of free thiols per antibody by dividing the thiol concentration adjusted for the dilution factor by the antibody concentration.

[0300] Example 14: Measurement of Drug-Antibody Ratio (DAR) by UV To estimate the DAR, the UV absorbance ratio (R) of the ADC is measured at 248 nm and 280 nm in a quartz cuvette: R = (absorbance at 248 nm) / (absorbance at 280 nm).

[0301] Calculate the DAR by applying the R value to the following formula: DAR = (21 x R - 9) / (1.615 - 0.1425 x R)

[0302] Example 15: Measurement of Drug-Antibody Ratio (DAR) by HIC-HPLC Another method that can be used to estimate the DAR of an ADC is by using HIC-HPLC analysis. Briefly, a sample of the ADC and unconjugated antibody is injected into the HPLC. The peaks observed at different retention times correspond to different drug-antibody ratios. The HPLC retention times and peak areas are analyzed in a spreadsheet, arbitrarily setting the DAR of the peaks to 1-8 for IgG1 and 1-10 for IgG2. The time between the peaks is used in combination with the analyst trying to fit these peaks to integers corresponding to a particular number of drugs conjugated to the antibody. This process is complicated by the fact that there are multiple different ADCs at each DAR. For example, for a DAR of 1, there are 8 possible ACDs (maleimide conjugation sites). In general, the difference in retention time between different DARs is greater than the difference in retention time in a group of ACDs with the same DAR. After the analyst sets the DAR for each peak, the peak areas are summed and the contribution from each DAR peak area is calculated. The average DAR of the sample is then determined based on all the peaks.

[0303] Drug-to-antibody ratio (DAR) and drug loading distribution by hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography, Jun Ouyan, Methods Mol. Biol. 2013;1045:275-83.

[0304] Example 16. Measuring target cell killing by MNC2-ADC or MN20A10-ADC using the xCELLigence assay Assays were performed on an xCELLigence RTCA MP instrument (Agilent) to evaluate real-time killing of target cancer cells, including breast cancer cells (T47D), non-small cell lung cancer cells (NCI-H1975), and pancreatic cancer cells (HPAF II). Target cancer cells were seeded at a density of 5000 cells / well (100 μL of 50,000 cells / mL stock) in multielectrode well plates and cultured for 24 h in a 37 °C / 5% CO2 incubator. mAb MNC2 ADC or mAb MN20A10-ADC were prepared as 2X solutions in the corresponding growth medium, and 100 μL of each concentration was added to the target cells. As a positive control (100% cell killing), cells were treated with 1% Triton or 1 μM Taxol instead of ADC. After 40–120 h of incubation in a 37 °C / 5% CO2 incubator, cell killing was evaluated. The assay measures impedance in real time: When attached cancer cells die and fall off the electrode surface, the impedance (insulation) decreases.

[0305] MNC2-ADC and MN20A10-ADC were assayed for their ability to kill both low and high MUC1* expressing cells by monitoring killing in real time with the xCELLigence instrument. In the xCelligence system, adherent target cancer cells are plated on an electrode array 96-well plate. The adherent cells insulate the electrodes and increase the impedance. The number of attached cancer cells is directly proportional to the impedance. Antibodies and antibody-drug conjugates are much smaller and do not contribute significantly to the impedance. Thus, an increase in impedance reflects cancer cell proliferation, and a decrease in impedance reflects cancer cell killing.

[0306] The breast cancer cell line T47D-wt expresses low to low-moderate levels of MUC1*. The pancreatic cancer cell line HPAF II-wt expresses even lower levels of MUC1*, and the lung cancer cell line NCI-H1975 expresses even lower levels of MUC1*. As shown in Figure 24, Figure 26, and Figure 27, these cancer cells expressing low levels of MUC1*, consistent with early stage cancer cells, are effectively killed by MNC2-ADC and MN20A10-ADC when administered at mid-nanomolar doses. The breast cancer cell lines T47D-MUC1* and HPAF II-MUC1* have been engineered to express high levels of MUC1*, consistent with late stage cancer. As shown in Figure 25 and Figure 28, cancer cells expressing high levels of MUC1* are completely killed at very low nanomolar doses. Figures 49A-49D show the killing potency of MNC2-MMAE, MN20A10-MMAE, MNC2-MMAF, and MN20A10-MMAF against T47D breast cancer cells expressing low to moderate levels of MUC1*. As can be seen, both anti-MUC1* antibodies MNC2 and MN20A10 effectively kill target cells when administered at a concentration of 500 nM within a 40 hour time frame. As can be seen in Figures 49E-49J, after 120 hours, potent killing is seen even at concentrations as low as 167 nM. Indeed, MN20A10-MMAE shows effective killing at 56 nM. Here, a comparison with MNC2-deruxtecan and MN20A10-deruxtecan shows comparable killing with MNC2-MMAE, MNC2-MMAF, MN20A10-MMAE, and MN20A10-MMAF at the same concentration, i.e., 167 nM.

[0307] In the next set of experiments, MNC2-ADC and MN20A10-ADC are tested for their ability to kill cancer cells expressing high levels of MUC1*. Since MUC1* is the growth factor receptor that drives the proliferation of these cells, it is expected that the higher the level of MUC1*, the more difficult it will be to kill the cells. Unexpectedly, the experiments show that the more MUC1* is expressed, the easier it is to kill the cells. Figures 50A-50D show that T47D breast cancer cells engineered to express high levels of MUC1*, indicative of late-stage cancer, are efficiently killed by MNC2-MMAE, MNC2-MMAF, and MN20A10-MMAE at concentrations as low as 19 nM and even as low as 6 nM. As illustrated in Figures N25E-N25J, MNC2-MMAE, MNC2-MMAF, and MNC2-deruxtecan showed nearly complete killing of cancer cells expressing high levels of MUC1* at concentrations as low as 6 nM when the experiment was continued for 120 h. MN20A10, which had fewer attachable MMAF and deruxtecan, showed cancer cell killing at higher concentrations between 19 nM and 167 nM.

[0308] Similar to breast cancer cells expressing low levels of MUC1*, NCI-H1975 non-small cell lung cancer cells expressing low levels of MUC1* became quiescent at 40 hours but died at 120 hours when treated with MNC2-MMAE, MNC2-MMAF, MNC2-deruxtecan, MN20A10-MMAE, or MN20A10-MMAF (Figures 51A-I). HPAF II-wt pancreatic cancer cells, which express low levels of MUC1* but at higher levels than H1975 lung cancer cells, were killed by MNC2-MMAE, MNC2-MMAF, MN20A10-MMAE, and MN20A10-MMAF at concentrations between 167 nM and 500 nM at 40 hours, improving to death at 56 nM after 120 hours (Figures 52A-I). However, when pancreatic cells are engineered to express high levels of MUC1*, MNC2-ADC and MN20A10-ADC are much more potent at killing cancer cells: after approximately 40 hours, near complete killing of cancer cells is seen at concentrations between 167 nM and 56 nM, and after 120 hours, near complete killing is achieved at concentrations between 19 nM and 56 nM (Figures 53A-I).

[0309] The toxin Exatecan was then conjugated to MNC2 via a linker containing maleimidocaproyl, valine-citrulline, and para-aminobenzyl, as shown in Figures N4E-N4H, and conjugation was further performed in a solution containing propylene glycol MES pH 6.0 buffer.

[0310] As illustrated in Figures 65A-65D, tumor cells implanted in animals significantly increase MUC1* expression as the tumor develops from early to late stages. The implanted tumor cells are T47D-wt breast cancer cells, a cell line derived from a breast cancer patient who died decades ago. The cell line provided by ATCC has been propagated thousands, if not millions, of times. Essentially all T47D cells are identical. When cells are stained at day 0, they express more full-length MUC1 than MUC1*, with weaker staining intensity and fewer MUC1* receptors, indicating early stage cancer. It can be easily recognized that 62 days after tumor implantation, which corresponds to about 7 years in human time, the expression of MUC1* has changed dramatically in terms of the extent of expression and the intensity of expression from low to high in late stage tumors. In contrast, staining of serial sections of the tumor reveals the expression of full-length MUC1, which is expected due to cleavage to MUC1* after surface expression. However, the intensity of staining did not increase, indicating that the majority of expressed MUC1 was cleaved into the growth factor receptor form MUC1* in late-stage tumors.

[0311] As known to cancer clinicians and pathologists, tumors that develop in patients are not homogenous populations of single clones, as cell lines are.Actual tumors are somewhat heterogeneous in that they are composed of low antigen expressing cells and high antigen expressing cells.However, it is also known that early cancers are characterized by the majority of cells being low expressers, while late cancers are characterized by the majority of cells being high expressers.

[0312] Example 17. Measuring target cell killing by MNC2-ADC or MN20A10-ADC using the PrestoBlue Live / Dead Cell Assay Another method to measure the killing activity of mAb MNC2-ADC and mAb MN20A10-ADC against target cancer cells, including T47D breast cancer cells, NCI-H1975 non-small cell lung cancer cells, and HPAF II pancreatic cancer cells, is to use the PrestoBlue live / dead cell assay (ThermoFisher). Target cancer cells were seeded at a density of 5000 cells / well (100 μL of 50,000 cells / mL stock) in black-walled, clear-bottom 96-well tissue culture plates and incubated overnight in a 37°C / 5% CO2 incubator. MNC2 ADC and MN20A10 ADC were prepared as 2X solutions in the corresponding cancer cell growth medium, and 100 μL of each concentration was added to the target cancer cells. As a positive control (100% killing), cells were treated with 1 μM taxol instead of the ADC. After 72-120 h of incubation in a 37°C / 5% CO2 incubator, cell viability was measured by fluorescence using the PrestoBlue HS assay (ThermoFisher). Briefly, after the incubation period, 20 μL of PrestoBlue HS solution was added. Fluorescence (Ex560nm / Em590nm) was recorded using a Tecan plate reader after 30-90 min of incubation. Viability was determined by PrestoBlue HS staining and normalized to 1 μM Taxol-treated cells.

[0313] Normalized fluorescence values ​​were imported into the graphing program Sigma Plot and data were plotted as MNC2-ADC or MN20A10-ADC concentration versus normalized fluorescence signal and curve-fitted using the four-parameter Hill equation: f=y0+a*x^b / (c^b+x^b).

[0314] IC50 values ​​are calculated from the fitted data for the killing potency of MNC2-ADC and MN20A10-ADC against target cancer cells and are reported in nM.

[0315] To visually assess target cell killing or changes in cancer cell morphology, bright-field images of target cancer cells, including T47D breast cancer cells, NCI-H1975 non-small cell lung cancer cells, and HPAF II pancreatic cancer cells, were taken at 4x or 20x magnification under an Olympus IX-71 microscope after incubation with the indicated concentrations of MNC2-ADC or MN20A10-ADC for 120 h.

[0316] Figures 46A-48N show magnified images of various types of cancer cells after treatment with either MNC2-ADC or MN20A10-ADC. The killing effect can be easily recognized as a significant reduction in cell number, a change in cell morphology from a characteristic flattened to a small rounded morphology, and floating of cells. In contrast, control wells showed a confluent monolayer of compact cells with a normal flattened morphology, and no floating dead cells were observed. Figures 46A-46C and Figures 46G-46F show T47D-MUC1* breast cancer cells treated with MNC2-MMAE. Figures 46D-46F and Figures 46G-46J show T47D-MUC1* breast cancer cells treated with MNC2-deruxtecan. Figures 47A-47B show DU145 hormone-resistant prostate cancer cells treated with MNC2-MMAE. Figures 47C-47D show DU145 hormone-resistant prostate cancer cells treated with MNC2-deruxtecan. Figures 48A-48B and Figures 48G-48H show T47D-MUC1* breast cancer cells treated with MN20A10-MMAE. Figures 48C-48D and Figures 48I-48J show T47D-MUC1* breast cancer cells treated with MN20A10-deruxtecan. Figures 48K-48L show DU145 hormone-resistant prostate cancer cells treated with MN20A10-MMAE. Figures 48M-48N show DU145 hormone-resistant prostate cancer cells treated with MN20A10-deruxtecan. As is visually apparent, the killing effects of anti-MUC1*-ADCs shown in Figures 46A-48N are consistent with killing measured by flow cytometry shown in Figures 38A-45D.

[0317] Example 18. Animal Study: Measurement of Target Cell Killing by MNC2-ADC or MN20A10-ADC In Vivo In vivo experiments were performed to measure targeted cancer cell killing in xenografts by MNC2-ADC or MN20A10-ADC.

[0318] For breast cancer xenografts, female NOD / SCID mice (18–22 g body weight, 6–8 weeks old, Charles River Laboratories) previously implanted with 90-day release estrogen pellets (Innovative Research Laboratories) were subcutaneously injected with 1 million luciferase-mCherry-positive T47D breast cancer cells expressing intermediate to high levels of MUC1* (ATCC) using BD 26G Insulin Syringes with Detachable Needles (Fisher cat: 329652) or BD 28G Lo-Dose U-100 Insulin Syringes (Fisher cat: 329461) under isoflurane anesthesia (ISOTHESIA, 250 mL (HENRY SCHEN, NDC: 11695-6776-2)).

[0319] For lung or pancreatic cancer xenografts, female NU / NU mice (body weight 18–22 g, 6–8 weeks old, Charles River Laboratories) were subcutaneously injected with 1 million luciferase-mCherry-positive NCI-H1975 non-small cell lung cancer cells (ATCC) expressing low-to-moderate levels of MUC1* or 1 million luciferase-mCherry-positive HPAF II pancreatic cancer cells (ATCC) under isoflurane anesthesia (ISOTHESIA, 250 mL (HENRY SCHEN, NDC: 11695-6776-2)).

[0320] Four to six days after xenografting, mice were injected intraperitoneally with 150ul, 30mg / mL D-luciferin (XenoLight™ D-luciferin potassium salt, PerkinElmer P / N122799) in the nape of the neck and then placed under isoflurane anesthesia before bioluminescence images were acquired using a Xenogen IVIS-Spectrum system (Perkin Elmer). Group selection was performed to distribute xenografted mice evenly to ensure that mock-treated and ADC-treated groups had comparable initial tumor sizes.

[0321] On days 5–7 after xenotransplantation, mice were injected intraperitoneally with phosphate-buffered saline (PBS) or 5–10 mg / kg MNC2-ADC or 5–10 mg / kg MN20A10-ADC. Mice were then given weekly injections of MNC2-ADC or MN20A10-ADC for a total of four injections.

[0322] Tumor growth was measured noninvasively in real time by weekly bioluminescence imaging, and data were graphed as radiance (photons / sec) as a function of days after tumor implantation.

[0323] Some measurements of tumor growth involved caliper measurements of tumors in mice. The two longest perpendicular axes in the x / y plane of each xenograft tumor were measured to the nearest 0.1 mm by two independent observers. Depth was assumed to correspond to the shortest of the perpendicular axes, defined as y. Measurements were performed with digital calipers while the mice were conscious and were calculated using the following formula: xenograft volume = xy 2 / 2.

[0324] Humane criteria for euthanasia were adhered to (per IACUC policy) if tumor burden reached or exceeded 20 mm in diameter, if tumor ulceration or tumor location prevented normal ambulation, feeding / drinking / or excretion, if body condition score was 2, or if weight loss of 15% or more from pre-xenotransplant weight occurred.

[0325] In vivo experiments were also performed to evaluate the killing potency of MNC2-ADC and MN20A10-ADC in immunocompromised mice implanted with several different types of human cancer. In all cases, the human cancer cell lines were engineered to express mCherry and also luciferase, allowing for measurement of tumors by bioluminescence. To optically measure tumor volume, animals are injected with luciferin, a luciferase substrate, and bioluminescence is photographed with an IVIS instrument within 10 minutes of luciferin injection. Luciferase expression levels vary widely in some cases, making it difficult to compare tumor size across different cell lines. In those cases, caliper measurements of tumors were also performed to allow comparison of tumor size between cell lines. Additionally, in those cases, animals were photographed to allow for visual comparison of tumor size between cell lines.

[0326] In one experiment, MNC2-MMAE was administered to female NOD / SCID / GAMMA (NSG) mice with 90-day estrogen-releasing pellets and then implanted with either 1M T47D-wt breast cancer cells expressing low to moderate levels of MUC1*, indicative of early-stage cancer, or 1M T47D-MUC1* cells engineered to express high levels of MUC1*, indicative of late-stage cancer. Tumors were allowed to engraft for 7 days before the first injection of MNC2-MMAE at a dose of 5 mg / kg. On days 14 and 20, the dose was increased to 10 mg / kg. First, T47D-wt tumors expressing low levels of MUC1* were implanted into the mice. Comparing the control mice in FIG. 57A with the MNC2-MMAE-treated mice in FIG. 57B, it can be easily seen that the treated mice survived with little to no tumor burden until day 56, when the experiment was terminated. In contrast, tumors in untreated control mice continued to grow and mice had to be sacrificed on day 56 due to excessive tumor burden. Next, mice implanted with T47D-MUC1* tumors, which express high levels of MUC1*, were reexamined. Comparing the bioluminescence of control animals implanted with T47D-wt (Figure 57A and Figure 57E) versus animals implanted with T47D-MUC1* (Figure 57C and Figure 57F), the T47D-MUC1* line expresses slightly less luciferase than the T47D-wt cell line. However, visual inspection of the tumors showed that tumor size was comparable between the two cell lines. MNC2-MMAE was administered to animals bearing T47D-MUC1* tumors according to an identical schedule as animals bearing T47D-wt tumors. However, in Figure 57D, it is clear that the MNC2-MMAE treated animals were tumor free from day 34 until the end of the experiment on day 56, when the control mice had excessive tumor burden and had to be sacrificed. This same experiment was performed in parallel, but animals were administered MN20A10-MMAE (Figures 58A-58F). The MN20A10-MMAE results follow the same trend as the MNC2-MMAE results, except that in this experiment MNC2-MMAE is more effective than MN20A10. One possible explanation for this discrepancy is the difference in the number of toxins conjugated to the MNC2 antibody (DAR 3.85) versus MN20A10 (DAR 2.96).

[0327] In another experiment, female nu / nu mice were implanted with 1M human NCI-H1975 non-small cell lung cancer cells expressing low to moderate levels of MUC1* indicative of early stage cancer. On days 7 and 14 after tumor implantation, animals were injected with 5 mg / kg MNC2-MMAE. On day 19, the dose was increased to 10 mg / kg. Bioluminescence photographs clearly show that increasing the dose of MNC2-MMAE to 10 mg / kg on days 19 and 27 increased tumor cell killing (Figures 59A-59C). The efficacy of MNC2-MMAE treatment can be assessed by comparing the tumor weights of sacrificed animals in the control group versus the tumor weights of the treated groups. Control animals had to be sacrificed on day 20 after implantation due to life-threatening tumor size, with tumor weights of 0.82, 0.3 grams, 0.49 grams, 0.41 grams, and 0.5 grams. In contrast, animals in the treatment group had to be sacrificed on day 54 weighing 0.28 grams and 0.4 grams. Two mice in the treatment group remained tumor-free on day 67, when the experiment was arbitrarily terminated. Results from a parallel experiment in which animals were treated with MN20A10-MMAE (Figures 60A-60D) showed the same trends, although MN20A10-MMAE was less effective than MNC2-MMAE, likely due to the lower DAR of MN20A10-MMAE. Nevertheless, the treatment suppressed tumor growth, as seen in the bioluminescence photographs and the weight of the tumor excised from the control animal, which had to be sacrificed on day 26. Tumor weights ranged from 2.2 grams to 0.65 grams. The graph in Figure 60D shows that tumor volume dropped dramatically after the dose was increased to 10 mg / kg on day 19. The data suggests that if the dose had been higher at the start, tumor growth would have decreased more dramatically.

[0328] In yet another experiment, MNC2-MMAE was administered to female nu / nu (NSG) mice implanted with either 0.5 M HPAF II-wt pancreatic cancer cells expressing low to moderate levels of MUC1*, indicative of early-stage cancer, or 0.5 M HPAF II-MUC1* pancreatic cancer cells engineered to express high levels of MUC1*, indicative of late-stage cancer. After allowing tumors to engraft for 5 days, MNC2-MMAE was injected three times at a dose of 10 mg / kg on days 5, 12, and 19. First, mice implanted with HPAF II-wt tumors expressing low levels of MUC1* were tested. Comparing the control mouse in FIG. 61A with the MNC2-MMAE-treated mouse in FIG. 61B, it is easy to see that on day 18, the treated mouse had a smaller tumor than the control mouse. The IVIS instrument did not function properly on day 25, resulting in an artificially low reading in the control animals. For this reason, caliper measurements were also performed on day 25. As can be seen in FIG. 61H, the mean tumor volumes measured by calipers differ significantly between control animals and treated animals implanted with HPAF II-wt tumors.

[0329] Next, mice implanted with T47D-MUC1* tumors, which express high levels of MUC1*, were tested. Comparing the bioluminescence of control animals implanted with T47D-wt (Figure 57A and Figure 57E) versus animals implanted with T47D-MUC1* (Figure 57C and Figure 57F), the T47D-MUC1* line expresses slightly less luciferase than the T47D-wt cell line. However, visual inspection of the tumors showed that tumor size was comparable between the two cell lines. Animals bearing T47D-MUC1* tumors were administered MNC2-MMAE according to an identical schedule to animals bearing T47D-wt tumors. However, in Figure 57D, it is clear that the MNC2-MMAE-treated animals were tumor-free from day 34 until day 56, when the experiment was terminated, as the control mice had excessive tumor burden and had to be sacrificed.

[0330] Embodiment Embodiment 1. Formula (I):

[0331] [ka] wherein X is a compound capable of inhibiting topoisomerase I or a moiety derived from a compound capable of inhibiting tubulin formation, R is a binding moiety, L is a dipeptide or tripeptide or tetrapeptide linking moiety having Z attached to the N-terminus and R attached to the C-terminus, and [Ab] is an antibody comprising an anti-MUC1* binding domain comprising three heavy chain (HC) complementarity determining regions (CDRs): MUC1* HC-CDR1, MUC1* HC-CDR2, and MUC1* HC-CDR3, wherein MUC1* HC-CDR1, MUC1* HC-CDR2, and MUC1* HC-CDR3 of the MUC1* binding domain comprise an amino acid sequence selected from those set out in Table 1, and the MUC1* binding domain comprises three light chain (LC) complementarity determining regions (CDRs): MUC1* LC-CDR1, MUC1* LC-CDR2, and MUC1* a conjugate comprising a MUC1* LC-CDR3, wherein MUC1* binding domain MUC1* LC-CDR1, MUC1* LC-CDR2, and MUC1* LC-CDR3 comprise an amino acid sequence selected from those shown in Table 1, wherein Z is a conjugate moiety capable of forming a covalent bond with the sulfur atom of a cysteine ​​residue, and y is an integer from 1 to 10.

[0332] Embodiment 2. A conjugate according to embodiment 1, wherein L comprises valine and citrulline.

[0333] Embodiment 3. The conjugate of embodiment 1, wherein L comprises glycine and phenylalanine.

[0334] Embodiment 4. The conjugate of embodiment 1, wherein R comprises para-aminobenzyl.

[0335] Embodiment 5. R is

[0336] [ka] wherein * indicates the point of attachment of the X group.

[0337] Embodiment 6.R comprises the following:

[0338] [ka] 2. The conjugate of embodiment 1, comprising a moiety comprising the structure:

[0339] Embodiment 7.R comprises the following:

[0340] [ka] 2. The conjugate of embodiment 1, comprising a moiety comprising the structure:

[0341] In embodiment 8.L,

[0342] [ka] 2. The conjugate of embodiment 1, wherein the dipeptide linking moiety comprises the structure:

[0343] In embodiment 9.L,

[0344] [ka] 2. The conjugate of embodiment 1, wherein the tetrapeptide binding moiety comprises the structure:

[0345] The conjugate of embodiment 1, wherein embodiment 1.0X is MMAE or MMAF.

[0346] Embodiment 11. The conjugate of embodiment 1, wherein X is exatecan or Dxd.

[0347] Embodiment 12.R is

[0348] [ka] 2. The conjugate of embodiment 1, comprising a moiety comprising the structure:

[0349] Embodiment 13. R is

[0350] [ka] wherein * indicates the point of attachment of the X group and X is exatecan.

[0351] Embodiment 14. The conjugate of embodiment 1, wherein the antibody is of isotype IgG1 or IgG2.

[0352] Embodiment 15. The conjugate of embodiment 1, wherein the antibody is of isotype IgG2b.

[0353] Embodiment 16. The conjugate of embodiment 1, wherein the anti-MUC1* binding domain comprises a heavy chain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 38 or 44.

[0354] Embodiment 17. A conjugate according to embodiment 1, wherein the anti-MUC1* binding domain comprises a heavy chain variable domain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 39 or 45.

[0355] Embodiment 18. The conjugate of embodiment 1, wherein the anti-MUC1* binding domain comprises a light chain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 41 or 47.

[0356] Embodiment 19. A conjugate according to embodiment 1, wherein the anti-MUC1* binding domain comprises a light chain variable domain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 42 or 48.

[0357] Embodiment 20. A conjugate according to embodiment 1, wherein the anti-MUC1* binding domain comprises a single chain variable fragment (scFv) comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 129 or 130.

[0358] Embodiment 21. An antibody conjugate comprising an antibody comprising an anti-MUC1* binding domain, wherein the antibody is conjugated to a payload via a maleimide-cysteine ​​bond, the payload comprising a linker and a cytotoxic compound, wherein the cytotoxic compound comprises a tubulin inhibitor or a topoisomerase I inhibitor.

[0359] Embodiment 22. The antibody conjugate of embodiment 21, wherein the linker comprises a valine.

[0360] Embodiment 23. The antibody conjugate of embodiment 21, wherein the linker comprises citrulline.

[0361] Embodiment 24. The antibody conjugate of embodiment 21, wherein the linker comprises valine and citrulline.

[0362] Embodiment 25. The linker is

[0363] [ka] 22. The antibody conjugate of embodiment 21, wherein the dipeptide binding moiety comprises the structure:

[0364] Embodiment 26 The antibody conjugate of embodiment 21, wherein the linker comprises at least one glycine.

[0365] Embodiment 27. The antibody conjugate of embodiment 21, wherein the linker comprises at least one glycine and phenylalanine.

[0366] Embodiment 28. The linker is

[0367] [ka] 22. The antibody conjugate of embodiment 21, comprising the structure:

[0368] Embodiment 29. The antibody conjugate of embodiment 21, wherein the linker comprises para-aminobenzyl.

[0369] Embodiment 30. The linker is

[0370] [ka] 22. The antibody conjugate of embodiment 21, comprising a group of the structure:

[0371] Embodiment 31. The linker is

[0372] [ka] 22. The antibody conjugate of embodiment 21, comprising a group of the structure:

[0373] Embodiment 32. The antibody conjugate of embodiment 21, wherein the tubulin inhibitor is MMAE or MMAF.

[0374] Embodiment 33 The antibody conjugate of embodiment 21, wherein the topoisomerase I inhibitor is exatecan or deruxtecan, or a derivative thereof.

[0375] Embodiment 34. The linker is

[0376] [ka] 22. The antibody conjugate of embodiment 21, comprising a group comprising the structure:

[0377] Embodiment 35. The linker is

[0378] [ka] 22. The antibody conjugate of embodiment 21, comprising a group comprising the structure:

[0379] Embodiment 36. The structure provided below:

[0380] [ka] wherein n is 1 to 10.

[0381] Embodiment 37. The structure provided below:

[0382] [ka] wherein n is 1 to 10.

[0383] Embodiment 38. The antibody conjugate of embodiment 21, wherein the antibody isotype is IgG1 or IgG2.

[0384] Embodiment 39. The antibody conjugate of embodiment 21, wherein the antibody isotype is IgG2b.

[0385] Embodiment 40. The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least two payloads.

[0386] Embodiment 41 The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least three payloads.

[0387] Embodiment 42. The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least four payloads.

[0388] Embodiment 43. The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least five payloads.

[0389] Embodiment 44. The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least six payloads.

[0390] Embodiment 45. The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least seven payloads.

[0391] Embodiment 46 The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least eight payloads.

[0392] Embodiment 47. The antibody conjugate of embodiment 21, wherein the anti-MUC1* binding domain comprises three light chain (LC) complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3 of the MUC1* binding domain comprise an amino acid sequence selected from those shown in Table 1, and at least one of LC-CDR1, LC-CDR2, and LC-CDR3 comprises 0 to 2 amino acid modifications.

[0393] Embodiment 48. The antibody conjugate of embodiment 21, wherein the anti-MUC1* binding domain comprises three heavy chain (HC) complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and HC-CDR1, HC-CDR2, and HC-CDR3 of the MUC1* binding domain comprise an amino acid sequence selected from those shown in Table 1, and at least one of HC-CDR1, HC-CDR2, and HC-CDR3 comprises 0 to 2 amino acid modifications.

[0394] Embodiment 49. The antibody conjugate of embodiment 21, wherein the anti-MUC1* binding domain comprises a heavy chain variable domain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence shown in Table 2.

[0395] Embodiment 50. The antibody conjugate of embodiment 21, wherein the anti-MUC1* binding domain comprises a light chain variable domain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence shown in Table 2.

[0396] Embodiment 51. The antibody conjugate of embodiment 21, wherein the anti-MUC1* binding domain comprises a single chain variable fragment (scFv) comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence shown in Table 3.

[0397] Embodiment 52. An antibody comprising a MUC1* binding domain and a CD3 binding domain, wherein the anti-MUC1* binding domain comprises three heavy chain (HC) complementarity determining regions (CDRs): MUC1* HC-CDR1, MUC1* HC-CDR2, and MUC1* HC-CDR3, wherein MUC1* HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 1, MUC1* HC-CDR2 comprises the amino acid sequence of SEQ ID NO: 2, and MUC1* HC-CDR3 comprises the amino acid sequence of SEQ ID NO: 3, and the MUC1* binding domain comprises three light chain (LC) complementarity determining regions (CDRs): MUC1* LC-CDR1, MUC1* LC-CDR2, and MUC1* LC-CDR3, wherein MUC1* LC-CDR1 comprises the amino acid sequence of SEQ ID NO: 13, and MUC1* LC-CDR2 comprises the amino acid sequence of SEQ ID NO: 14, and an antibody having a CD3 binding domain comprising an amino acid sequence selected from those shown in Table 4, and a CD3 binding domain comprising three heavy chain (HC) complementarity determining regions (CDRs): CD3 HC-CDR1, CD3 HC-CDR2, and CD3 HC-CDR3, wherein CD3 HC-CDR1, CD3 HC-CDR2, and CD3 HC-CDR3 of the CD3 binding domain comprise an amino acid sequence selected from those shown in Table 2, and wherein the CD3 binding domain comprises three light chain (LC) complementarity determining regions (CDRs): CD3 LC-CDR1, CD3 LC-CDR2, and CD3 LC-CDR3, wherein CD3 LC-CDR1, CD3 LC-CDR2, and CD3 LC-CDR3 of the CD3 binding domain comprise an amino acid sequence selected from those shown in Table 4.

[0398] Embodiment 53. The antibody of embodiment 52, comprising an Fc domain.

[0399] Embodiment 54. The antibody of embodiment 52, wherein the Fc domain is a heterodimeric Fc domain.

[0400] Embodiment 55. The antibody of embodiment 52, wherein the heterodimeric Fc domain comprises a knob chain and a hole chain and forms a knob-in-hole (KiH) structure.

[0401] Embodiment 56 The antibody of embodiment 55, wherein the knob chain comprises a sequence having at least about 95% identity to a sequence selected from SEQ ID NOs: 121, 122, 123, or 124.

[0402] Embodiment 57. The antibody of embodiment 55, wherein the hole chain comprises a sequence having at least about 95% identity to a sequence selected from SEQ ID NOs: 125, 126, 127, or 128.

[0403] Embodiment 58. The antibody of embodiment 52, wherein the anti-MUC1* binding domain comprises a heavy chain variable domain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from Table 2.

[0404] Embodiment 59. The antibody of embodiment 52, wherein the anti-MUC1* binding domain comprises a light chain variable domain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from Table 2.

[0405] Embodiment 60. The antibody of embodiment 52, wherein the anti-MUC1* binding domain comprises a single chain variable fragment (scFv) comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from Table 3.

[0406] Embodiment 61. The antibody of embodiment 52, wherein the CD3 binding domain comprises a heavy chain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 26 or 31.

[0407] Embodiment 62. The antibody of embodiment 52, wherein the CD3 binding domain comprises a light chain comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 29 or 35.

[0408] Embodiment 63. The antibody of embodiment 52, wherein the CD3 binding domain comprises a single chain variable fragment (scFv) comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NO: 131 or 132.

[0409] Embodiment 64. The antibody of embodiment 52, comprising a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from SEQ ID NOs: 50, 52, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, or 114.

[0410] Embodiment 65. A method for treating cancer, comprising administering to a subject in need of such treatment an antibody or antibody conjugate according to any one of embodiments 1 to 64.

[0411] Embodiment 66 The method of embodiment 65, wherein the cancer expresses MUC1*.

[0412] Embodiment 67. The method of embodiment 65, wherein the cancer is breast cancer, colon cancer, prostate cancer, pancreatic cancer, or lung cancer.

Claims

1. Equation (I): 【Chemistry 1】 It is a conjugate of, During the ceremony, [Ab] is Three heavy chain (HC) complementarity-determining regions (CDRs): MUC1*HC-CDR1, MUC1*HC-CDR2, and MUC1*HC-CDR3, Three light chain (LC) complementarity-determining regions (CDRs): MUC1*LC-CDR1, MUC1*LC-CDR2, and MUC1*LC-CDR3 It is an antibody containing an anti-MUC1* binding domain, (a) The MUC1*HC-CDR1 contains the amino acid sequence of SEQ ID NO: 1, the MUC1*HC-CDR2 contains the amino acid sequence of SEQ ID NO: 2, the MUC1*HC-CDR3 contains the amino acid sequence of SEQ ID NO: 3, the MUC1*LC-CDR1 contains the amino acid sequence of SEQ ID NO: 13, the MUC1*LC-CDR2 contains the amino acid sequence of SEQ ID NO: 14, the MUC1*LC-CDR3 contains the amino acid sequence of SEQ ID NO: 15, or (b) The MUC1*HC-CDR1 contains the amino acid sequence of SEQ ID NO: 4, the MUC1*HC-CDR2 contains the amino acid sequence of SEQ ID NO: 5, the MUC1*HC-CDR3 contains the amino acid sequence of SEQ ID NO: 6, the MUC1*LC-CDR1 contains the amino acid sequence of SEQ ID NO: 16, the MUC1*LC-CDR2 contains the amino acid sequence of SEQ ID NO: 17, and the MUC1*LC-CDR3 contains the amino acid sequence of SEQ ID NO:

18. Z is a conjugate portion that forms a covalent bond with the sulfur atom of the cysteine ​​residue of the antibody, L is a dipeptide, tripeptide, or tetrapeptide bond, Z is attached to the N-terminus of L, and R is attached to the C-terminus of L. R is the bonding part, X is selected from exatecan, Dxd, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF), where Dxd is -O-CH2CO-exatecan, and, y is a conjugate, an integer between 1 and 8.

2. The antibody is a. A heavy chain containing a sequence having at least 90% identity with the amino acid sequence of Sequence ID No. 39, and A light chain containing a sequence having at least 90% identity with the amino acid sequence of Sequence ID No. 42, or b. A heavy chain containing a sequence having at least 90% identity with the amino acid sequence of Sequence ID No. 45, and A light chain containing a sequence having at least 90% identity with the amino acid sequence of Sequence ID No.

48. The conjugate according to claim 1, including the following:

3. The conjugate according to claim 1, wherein y is at least 3.

4. The conjugate according to claim 1, wherein L comprises two, three, or four amino acids selected from the group comprising valine, citrulline, glycine, alanine, isoleucine, methionine, tryptophan, serine, threonine, asparagine, glutamine, cysteine, tyrosine, leucine, tryptophan, proline, lysine, arginine, histidine, aspartate, glutamate, and phenylalanine.

5. L is a. 【Chemistry 2】 A dipeptide bond portion containing, or b. 【Transformation 3】 The conjugate according to claim 4, wherein the tetrapeptide bond portion contains the tetrapeptide bond portion.

6. R is a. 【Chemistry 4】 b. 【Transformation 5】 or c. 【Transformation 6】 The conjugate according to claim 1, comprising, where * indicates a bond site of an X group.

7. The conjugate according to claim 1, wherein X is exatecan or Dxd.

8. The conjugate according to claim 1, wherein X is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). 【Request Item 9】 【Chemistry 7】 The conjugate according to claim 1, including the following:

10. The MUC1 * HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 4, The aforementioned MUC1*HC-CDR2 contains the amino acid sequence of SEQ ID NO: 5, The aforementioned MUC1*HC-CDR3 contains the amino acid sequence of SEQ ID NO: 6, The aforementioned MUC1 * LC-CDR1 contains the amino acid sequence of SEQ ID NO: 16, The aforementioned MUC1 * LC-CDR2 contains the amino acid sequence of SEQ ID NO: 17, and The conjugate according to claim 9, wherein the MUC1 * LC-CDR3 comprises the amino acid sequence of SEQ ID NO:

18.

11. The conjugate according to claim 9, wherein Ab comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 38 and a light chain consisting of the amino acid sequence of SEQ ID NO:

41.

12. The conjugate according to claim 9, wherein Ab comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 44 and a light chain consisting of the amino acid sequence of SEQ ID NO:

47.

13. The conjugate according to claim 12, wherein y is at least 3. 【Request Item 14】 【Chemistry 8】 Includes, The conjugate according to claim 1, wherein Ab comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 38 and a light chain consisting of the amino acid sequence of SEQ ID NO:

41. 【Request Item 15】 【Chemistry 9】 Includes, Ab is, A heavy chain consisting of the amino acid sequence of SEQ ID NO: 38 and a light chain consisting of the amino acid sequence of SEQ ID NO: 41, or A heavy chain consisting of the amino acid sequence of SEQ ID NO: 44 and a light chain consisting of the amino acid sequence of SEQ ID NO: 47 The conjugate according to claim 1, including the following: 【Request Item 16】 【Chemistry 10】 The conjugate according to claim 1, including the following:

17. The aforementioned antibody a. A heavy chain containing the amino acid sequence of SEQ ID NO: 39 and a light chain containing the amino acid sequence of SEQ ID NO: 42, or b. A heavy chain containing the amino acid sequence of SEQ ID NO: 45 and a light chain containing the amino acid sequence of SEQ ID NO: 48 The conjugate according to claim 1, including the following:

18. Use of the conjugate according to claim 1 in the manufacture of a drug for use in a method of treating cancer, wherein the method comprises the step of administering the conjugate to a subject.

19. The use according to claim 18, wherein the cancer expresses MUC1*.

20. The use according to claim 19, wherein the cancer includes breast cancer, colon cancer, prostate cancer, pancreatic cancer, or lung cancer.

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