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 comprising a binding domain of MUC1 * and a binding domain to CD3. The disclosure also provides compositions comprising the antibodies and methods for treating diseases and disorders, such as cancer.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 330,277, filed on April 12, 2022, the entire contents of which are hereby incorporated by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in ASCII form and is hereby incorporated by reference in its entirety. The ASCII copy was created on April 4, 2023, is named 56699-751.601_SL.xml, and is 230 kilobytes in size.

[0005] Incorporation by reference

[0006] 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 herein by reference. If publications and patents or patent applications incorporated by reference conflict with the disclosure in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. Background Art

[0007] Provided herein are multispecific antibodies comprising a MUC1* binding domain and a CD3 binding domain. Also provided herein are antibody conjugates of the formula described herein comprising one or more moieties derived from a therapeutic agent (e.g., a topoisomerase I inhibitor, a microtubule formation inhibitor), and wherein the conjugate further comprises 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 can be used to treat a disease or disorder, e.g., a proliferative disease such as cancer. Also provided herein are uses and methods of using these multispecific antibodies and antibody conjugates for treating diseases and disorders. Summary of the invention

[0008] Disclosed herein is a conjugate of formula (I): [Ab]-[ZLRX] yFormula (I), 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 coupling moiety; L is a dipeptide, tripeptide or tetrapeptide linking moiety having Z bound to the N-terminus and R bound to the C-terminus; [Ab] is an antibody comprising an anti-MUC1* binding domain, the 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 R2 and MUC1*HC-CDR3 comprise an amino acid sequence selected from those specified in Table 1; wherein 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, MUC1*LC-CDR2 and MUC1*LC-CDR3 of the MUC1* binding domain comprise an amino acid sequence selected from those specified in Table 1; Z is a conjugated moiety capable of forming a covalent bond with the sulfur atom of a cysteine ​​residue; and wherein y is an integer from 1 to 10.

[0009] L may include valine and citrulline. L may include glycine and phenylalanine. R may include p-aminobenzyl. R may include structures including Where * represents the point of attachment of the X group. R may include a moiety comprising the structure Where * represents the point of attachment of the X group. R may include a moiety comprising the structure where * indicates the point of attachment of the X group.

[0010] L can be a structure containing L may be a dipeptide linking portion comprising the structure The tetrapeptide linker portion.

[0011] X can be MMAE or MMAF. X can be exatecan or Dxd. R can include the structure Wherein * represents the attachment point of the X group, and wherein X is Dxd. R may include structures wherein * represents the point of attachment of the X group, and wherein X is exatecan.

[0012] The antibody may be of isotype IgG1 or IgG2. The antibody may be of isotype IgG2b.

[0013] The anti-MUCl* binding domain can include 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. The anti-MUCl* binding domain can include 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.

[0014] The anti-MUCl* binding domain can include 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. The anti-MUCl* binding domain can include 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.

[0015] The anti-MUC1* binding domain can include 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.

[0016] Disclosed herein is 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, wherein the payload comprises a linker and a cytotoxic compound, wherein the cytotoxic compound comprises 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 a structure The dipeptide linking portion of the invention may include at least one glycine. The linker may include at least one glycine and a phenylalanine. The linker may include the structure The linker may comprise a p-aminobenzyl group. The linker may comprise a structural group Where * indicates the attachment point of the cytotoxic group. The linker may contain the structural group Where * indicates the attachment point of the cytotoxic group. The microtubule inhibitor can be MMAE or MMAF. The topoisomerase I inhibitor can be exatecan or derutecan, or a derivative thereof. The linker can include a structure wherein * represents the attachment point of the cytotoxic group, and wherein the topoisomerase I inhibitor is Dxd. The linker may include a structure wherein * indicates the point of attachment of the cytotoxic group, and wherein the topoisomerase I inhibitor is exatecan.

[0017] The antibody conjugate may comprise the structure provided below, wherein n is 1 to 10:

[0018] The antibody conjugate may comprise the structure provided below, wherein n is 1 to 10:

[0019] The antibody isotype may be IgG1 or IgG2. The antibody isotype may be IgG2b. 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) complementary determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3; wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the MUC1* binding domain may comprise an amino acid sequence selected from those specified in Table 1; and wherein 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; wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the MUC1* binding domain may comprise an amino acid sequence selected from those specified in Table 1; and wherein 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 include 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 the sequence specified in Table 2. The anti-MUCl* binding domain can include 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 the sequence set forth in Table 2. The anti-MUCl* binding domain can include 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 the sequence set forth in Table 3.

[0021] Single chain variable fragments (scFv) with sequences that are 98% or 99% identical.

[0022] Disclosed herein is an antibody comprising a MUC1* binding domain and a CD3 binding domain, wherein the 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, MUC1* HC-CDR2 may comprise the amino acid sequence of SEQ ID NO: 2, and 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, MUC1* LC-CDR2 may comprise the amino acid sequence of SEQ ID NO: 14, NO:14, and MUC1*LC-CDR3 may comprise the amino acid sequence of SEQ ID NO:15; wherein the CD3 binding domain may comprise 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 may comprise an amino acid sequence selected from those specified in Table 2; and wherein the CD3 binding domain may comprise 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 may comprise an amino acid sequence selected from those specified in Table 4.

[0023] 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, forming a knob-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 include 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 include 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 include 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 3.

[0024] The CD3 binding domain may include a single chain variable fragment (scFv) 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.

[0025] %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a heavy chain of an amino acid sequence selected from SEQ ID NO: 29 or 35. The CD3 binding domain may include a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a heavy chain of an amino acid sequence selected from SEQ ID NO: 29 or 35.

[0026] The CD3 binding domain may include 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: 131 or 132. The antibody may comprise a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical 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.

[0027] Disclosed herein is a method of treating cancer comprising administering to a subject in need thereof an antibody of any one of claims 1-64. In some embodiments, the cancer expresses MUC1*. The cancer can be breast cancer, colon cancer, prostate cancer, pancreatic cancer, or lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A-Figure 1P A photograph is shown of MUC1*-positive breast cancer cells T47D cultured with human T cells to which different concentrations of the bispecific antibody 20A10-OKT3-BiTE were added. 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 in the figure, the addition of the bispecific antibody mediates engagement of the T cells and cancer cells, shown here as T cell clustering, a hallmark of activation directed by the bispecific bridge between the T cell CD3 and the cancer cell MUC1*. Figure 1A The concentration of the bispecific antibody was 1,000 ng / mL. Figure 1B The concentration is 333ng / mL. Figure 1C The concentration is 111ng / mL. Figure 1D In the Figure 1E The concentration is 12.3 ng / mL. Figure 1F The concentration is 4.1 ng / mL. Figure 1G In the Figure 1H The concentration is 0.4ng / mL. Fig. 1I The concentration is 0.15 ng / mL. Figure 1J The concentration is 0.05ng / mL. Figure 1Kare control wells in which both T cells and cancer cells were present but no bispecific antibody was added. Figure 1L are control wells, in which only cancer cells are present. Figure 1M A cartoon depicting how the LDH cytotoxicity assay works is shown, where higher A490 measurements indicate higher cell killing. Figure 1N Shown is a graph of cell killing as a function of 20A10-OKT3 (anti-MUC1* / anti-CD3 bispecific antibody) concentration. Fig.1O Shown is a graph of secreted interferon-γ secreted by T cells as a function of added anti-MUC1* / anti-CD3 bispecific antibody, wherein the anti-MUC1* antibody is 20A10 and the anti-CD3 antibody is OKT3. Figure 1P Shown is a graph of secreted TNF-α secreted by T cells as a function of added anti-MUC1* / anti-CD3 bispecific antibody, wherein the anti-MUC1* antibody is 20A10 and the anti-CD3 antibody is OKT3.

[0029] Figure 2A-2P A photograph showing MUC1*-positive breast cancer cells T47D cultured with human T cells to which different concentrations of the bispecific antibody 20A10-12F6-BiTE were 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 in the figure, the addition of the bispecific antibody mediates the engagement of T cells and cancer cells, shown here as T cell clustering, a hallmark of activation directed by the bispecific bridge between T cell CD3 and cancer cell MUC1*. Figure 2A The concentration of the bispecific antibody was 1,000 ng / mL. Figure 2B The concentration is 333ng / mL. Figure 2C The concentration is 111ng / mL. Figure 2D In the Figure 2E The concentration is 12.3 ng / mL. Figure 2F The concentration is 4.1 ng / mL. Figure 2G In the Figure 2H The concentration is 0.4ng / mL. Fig.2I The concentration is 0.15 ng / mL. Figure 2J The concentration is 0.05ng / mL. Figure 2K are control wells in which both T cells and cancer cells were present but no bispecific antibody was added. Figure 2L are control wells, in which only cancer cells are present. Figure 2MA cartoon depicting how the LDH cytotoxicity assay works is shown, where higher A490 measurements indicate higher cell killing. Figure 2N Shown is a graph of cell killing as a function of 20A10-OKT3 (anti-MUC1* / anti-CD3 bispecific antibody) concentration. Fig.2O Shown is a graph of secreted interferon-γ secreted by T cells as a function of added anti-MUC1* / anti-CD3 bispecific antibody, wherein the anti-MUC1* antibody is 20A10 and the anti-CD3 antibody is 12F6. Figure 2P Shown is a graph of secreted TNF-α secreted by T cells as a function of added anti-MUC1* / anti-CD3 bispecific antibody, wherein the anti-MUC1* antibody is 20A10 and the anti-CD3 antibody is 12F6.

[0030] Figure 3A-Figure 3L A photograph showing HCT-MUC1* transduced cancer cells cultured with human T cells to which different concentrations of the bispecific antibody 20A10-OKT3-BiTE were added. 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 in the figure, the addition of the bispecific antibody mediates engagement of the T cells and cancer cells, shown here as cell aggregation. Figure 3A The concentration of the bispecific antibody was 1,000 ng / mL. Figure 3B The concentration is 333ng / mL. Figure 3C The concentration is 111ng / mL. Figure 3D In the Figure 3E The concentration is 12.3 ng / mL. Figure 3F The concentration is 4.1 ng / mL. Figure 3G In the Figure 3H The concentration is 0.4ng / mL. Fig. 3I The concentration is 0.15 ng / mL. Figure 3J The concentration is 0.05ng / mL. Figure 3K are control wells in which both T cells and cancer cells were present but no bispecific antibody was added. Figure 3L are control wells, in which only cancer cells were present.

[0031] Figure 4A-4LA photograph is shown of HCT-MUC1* transduced cancer cells cultured with human T cells to which different concentrations of the bispecific antibody 20A10-12F6-BiTE were 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 in the figure, the addition of the bispecific antibody mediates engagement of the T cells and cancer cells, as seen here with cell aggregation. Figure 4A The concentration of the bispecific antibody was 1,000 ng / mL. Figure 4B The concentration is 333ng / mL. Figure 4C The concentration is 111ng / mL. Figure 4D In the Figure 4E The concentration is 12.3 ng / mL. Figure 4F The concentration is 4.1 ng / mL. Figure 4G In the Figure 4H The concentration is 0.4ng / mL. Fig. 4I The concentration is 0.15 ng / mL. Figure 4J The concentration is 0.05ng / mL. Figure 4K are control wells in which both T cells and cancer cells were present but no bispecific antibody was added. Figure 4L are control wells in which bispecific antibodies have been added to cancer cells but no T cells are present.

[0032] Figure 5A-5F Shown is a photograph of HCT-WT cancer cells that are negative for MUC1 and MUC1*. The cancer cells have been incubated with the MNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently coupled to the deglycosylated MNC2, which binds to MUC1*. As can be seen in the figure, the addition of the MNC2-ADC did not affect the viability of these HCT, MUC1-negative cells. Figure 5A The concentration of MNC2-ADC was 100 nM. Figure 5B The concentration of MNC2-ADC was 10 nM. Figure 5C The concentration of MNC2-ADC was 0.1 nM. Figure 5D The concentration of MNC2-ADC was 0.01 nM. Figure 5E The concentration of MNC2-ADC was 0.001 nM. Fig. 5F In the assay, the concentration of MNC2-ADC was 0 nM.

[0033] Figure 6A-6GA photograph of HCT-MUC1* cancer cells is shown. These HCT cells have been transduced to express the target of the antibody MNC2, namely MUC1*. The cancer cells have been incubated with the MNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently coupled to the deglycosylated MNC2, which binds to MUC1*. As shown in the figure, the addition of MNC2-ADC at the highest concentration tested here, 100nM, induced cell agglutination, an indicator of cell death. Fig. 6A The concentration of MNC2-ADC was 100 nM. Figure 6B The concentration of MNC2-ADC was 10 nM. Figure 6C The concentration of MNC2-ADC was 1.0 nM. Fig.6D The concentration of MNC2-ADC was 0.1 nM. Fig. 6E The concentration of MNC2-ADC was 0.01 nM. Fig. 6F The concentration of MNC2-ADC was 0.001 nM. Figure 6G In the assay, the concentration of MNC2-ADC was 0 nM.

[0034] Figure 7A-7G Shown is a photograph of K562-WT cells, which are negative for MUC1 and MUC1*. The cells have been incubated with the MNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) is covalently coupled to the deglycosylated MNC2, which binds to MUC1*. As can be seen in the figure, the addition of the MNC2-ADC does not affect the viability of these MUC1-negative cells. Fig. 7A The concentration of MNC2-ADC was 100 nM. Figure 7B The concentration of MNC2-ADC was 10 nM. Figure 7C The concentration of MNC2-ADC was 1.0 nM. Fig.7D The concentration of MNC2-ADC was 0.1 nM. Fig. 7E The concentration of MNC2-ADC was 0.01 nM. Figure 7F The concentration of MNC2-ADC was 0.001. Figure 7G In the assay, the concentration of MNC2-ADC was 0 nM.

[0035] Figure 8A-8GA photograph of K562-MUC1* cells is shown. These MUC1* negative cells were transduced to express MUC1*. The cells have been incubated with MNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently coupled to the deglycosylated MNC2, which binds to MUC1*. As can be seen in the figure, the addition of MNC2-ADC at the higher concentrations tested here, 10nM and 100nM, induced cell agglutination, an indicator of cell death. Fig. 8A The concentration of MNC2-ADC was 100 nM. Figure 8B The concentration of MNC2-ADC was 10 nM. Figure 8C The concentration of MNC2-ADC was 1.0 nM. Fig.8D The concentration of MNC2-ADC was 0.1 nM. Fig. 8E The concentration of MNC2-ADC was 0.01 nM. Figure 8F The concentration of MNC2-ADC was 0.001 nM. Figure 8G In the assay, the concentration of MNC2-ADC was 0 nM.

[0036] Figure 9A-9B A graph of a cell viability assay is shown, in which PrestoBlue is used to measure cell death. The graph shows cell viability as a function of MNC2-ADC concentration, comparing the effects of MNC2-ADC on K562-WT cells and K562-MUC1* cells. It can be seen that at concentrations of 10nM and 100nM, the MNC2-ADC antibody only induces cell death in cells expressing MUC1*. Fig. 9B Cell viability as a function of MNC2-ADC concentration is shown, comparing the effects of MNC2-ADC on HCT-WT cells and HCT-MUC1* cells. It can be seen that at high concentrations, the MNC2-ADC antibody only induces cell death in cells expressing MUC1*.

[0037] Figures 10A-10G Shown is a photograph of T47D-WT breast cancer cells expressing both full-length MUC1 (to which MNC2 does not bind) and MUC1* (to which MNC2 binds). The cells have been incubated with the MNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently coupled to the deglycosylated MNC2, which binds to MUC1*. Fig. 10A The concentration of MNC2-ADC was 100 nM. Fig. 10B The concentration of MNC2-ADC was 10 nM. Fig. 10C The concentration of MNC2-ADC was 1.0 nM. Fig. 10DThe concentration of MNC2-ADC was 0.1 nM. Fig.10E The concentration of MNC2-ADC was 0.01 nM. Fig.10F The concentration of MNC2-ADC was 0.001. Figure 10G In the assay, the concentration of MNC2-ADC was 0 nM.

[0038] Figures 11A-11G Shown is a photograph of T47D-MUC1* cells that have been transduced to express more MUC1*. The cells have been incubated with the MNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) is covalently coupled to the deglycosylated MNC2, which binds to MUC1*. As can be seen in the figure, the addition of the MNC2-ADC at the higher concentrations tested here, 10nM and 100nM, induces cell agglutination, an indicator of cell death. Fig.11A The concentration of MNC2-ADC was 100 nM. Fig. 11B The concentration of MNC2-ADC was 10 nM. Fig. 11C The concentration of MNC2-ADC was 1.0 nM. Fig.11D The concentration of MNC2-ADC was 0.1 nM. Fig.11E The concentration of MNC2-ADC was 0.01 nM. Fig.11F The concentration of MNC2-ADC was 0.001. Fig.11G In the assay, the concentration of MNC2-ADC was 0 nM. Fig.12 A graph of a cell viability assay is shown, where PrestoBlue is used to detect dead cells. The graph shows cell viability as a function of MNC2-ADC concentration, comparing the effects of MNC2-ADC on T47D-WT cells and T47D-MUC1* cells. It can be seen that at concentrations of 10nM and 100nM, the MNC2-ADC antibody induces cell death in cells expressing MUC1*.

[0039] Fig.12 A graph of a cell viability assay is shown, in which PrestoBlue is 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 and T47D-MUC1* cells. It can be seen that at concentrations of 10nM and 100nM, the MNC2-ADC antibody induced cell death in cells expressing MUC1*.

[0040] Figure 13A-13JShown are photos of T47D-WT breast cancer cells that express both full-length MUC1 (to which MNC2 does not bind) and MUC1* (to which MNC2 binds). The cells have been incubated with the MNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) was covalently coupled to the deglycosylated MNC2, which binds to MUC1*. It can be seen that the MNC2-ADC antibody induces T47D-WT cell death at the highest concentration of 1000nM. Fig.13A The concentration of MNC2-ADC was 0 nM. Fig. 13B The concentration of MNC2-ADC was 0.1 nM. Fig. 13C The concentration of MNC2-ADC was 0.39 nM. Fig.13D The concentration of MNC2-ADC was 1.0 nM. Fig.13E The concentration of MNC2-ADC was 3.9 nM. Fig.13F The concentration of MNC2-ADC was 10 nM. Figure 13G The concentration of MNC2-ADC was 39 nM. Fig.13H The concentration of MNC2-ADC was 100 nM. Fig.13I The concentration of MNC2-ADC was 393 nM. Fig.13J In the assay, the concentration of MNC2-ADC was 1000 nM.

[0041] Figure 14A-14J Shown is a photograph of T47D-MUC1* cells that have been transduced to express more MUC1*. The cells have been incubated with the MNC2-ADC for 72 hours. In this particular case, the toxic MMAE (monomethyl auristatin) is covalently coupled to the deglycosylated MNC2, which binds to MUC1*. As shown in the figure, the addition of the MNC2-ADC at the higher concentrations tested here, 10nM, 39nM, 100nM, 393nM, and 1000nM, induced cell aggregation, an indicator of cell death. Fig.14A The concentration of MNC2-ADC was 0 nM. Fig. 14B The concentration of MNC2-ADC was 0.1 nM. Fig. 14C The concentration of MNC2-ADC was 0.39 nM. Fig.14D The concentration of MNC2-ADC was 1.0 nM. Fig.14E The concentration of MNC2-ADC was 3.9 nM. Fig.14F The concentration of MNC2-ADC was 10 nM. Figure 14G The concentration of MNC2-ADC was 39 nM. Fig.14H The concentration of MNC2-ADC was 100 nM. Fig.14I The concentration of MNC2-ADC was 393 nM. Fig.14J In the assay, the concentration of MNC2-ADC was 1000 nM.

[0042] Figure 15A-15B A graph of a cell viability assay is shown, in which PrestoBlue is used to measure cell death. The graph shows cell viability as a function of MNC2-ADC concentration, comparing the effects of MNC2-ADC on T47D-WT cells and T47D-MUC1* cells. It can be seen that at the highest concentration of 1000nM, the MNC2-ADC antibody induced T47D-WT cell death, while T47D-MUC1* cells were killed at concentrations of 10nM, 39nM, 100nM, 393nM, and 1000nM.

[0043] Figures 16A-16F A magnified photograph of cancer cells to which MNC2-ADC was added over a range of concentrations is shown. Here, the toxin conjugated to the antibody is MMAE. Fig.16A Photos of breast cancer cells T47D wild type with MNC2-ADC added in a concentration range of 500 ng / mL to 0.1 ng / mL are shown. As a control, no MNC2-ADC was added. Photos were taken 72 hours after MNC2-ADC was added to cancer cells. Fig. 16B Shown are photos of breast cancer cells T47D-MUC1*, meaning these cells are stably transfected with more MUC1* than naturally expressed, with MNC2-ADC added at concentrations ranging from 500ng / mL to 0.1ng / mL. As a control, no MNC2-ADC was added. The photos were taken 72 hours after MNC2-ADC was added to the cancer cells. Fig. 16C Photos of breast cancer cells T47D wild type are shown, where 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, the MNC2-ADC was removed after 16 hours and the medium was replaced. The photos were taken 72 hours after the MNC2-ADC was initially added to the cancer cells. Fig.16D Shown are photos of breast cancer cells T47D-MUC1*, meaning these cells are stably transfected with more MUC1* than naturally expressed, with MNC2-ADC added at concentrations ranging from 500ng / mL to 0.1ng / mL. As a control, no MNC2-ADC was added. In this case, the MNC2-ADC was removed after 16 hours and the media was replaced. The photos were taken 72 hours after the MNC2-ADC was initially added to the cancer cells. Fig.16EShown are magnified photographs of T47D-wt cells treated with 1 μM paclitaxel for 72 hours. Fig.16F Shown are magnified photographs of T47D-MUC1* cells treated with 1 μM paclitaxel for 72 hours.

[0044] Fig.17 A graph showing cancer cell killing as a function of the concentration of MNC2-ADC MMAE added to the cell culture medium for 72 hours or 16 hours of treatment is shown. In the latter case, the medium was changed to medium without MNC2-ADC after 16 hours, and the experiment was allowed to continue until 72 hours after the first addition of MNC2-ADC to cancer cells. Cell viability was measured using PrestoBlue. Cell death was normalized to paclitaxel added to a final concentration of 1 μM, while the percentage of live cells at 72 hours was normalized to 0% viability.

[0045] Figure 18A-18F Magnified photographs of cancer cells to which 20A10-ADC was added over a range of concentrations are shown. Here, the toxin conjugated to the antibody is MMAE. Fig.18A Photos of breast cancer cells T47D wild type with 20A10-ADC added at concentrations ranging from 500 ng / mL to 0.1 ng / mL are shown. As a control, no 20A10-ADC was added. Photos were taken 72 hours after 20A10-ADC was added to cancer cells. Fig.18B Shown are photos of breast cancer cells T47D-MUC1*, meaning these cells are stably transfected with more MUC1* than naturally expressed, with 20A10-ADC added at concentrations ranging from 500 ng / mL to 0.1 ng / mL. As a control, no 20A10-ADC was added. The photos were taken 72 hours after 20A10-ADC was added to the cancer cells. Fig.18C Shown are photos of breast cancer cells T47D wild type, where 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 photo was taken 72 hours after the initial addition of 20A10-ADC to the cancer cells. Fig.18D Shown are photos of breast cancer cells T47D-MUC1*, meaning these cells are stably transfected with more MUC1* than they naturally express, with 20A10-ADC 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 media was replaced. The photo was taken 72 hours after 20A10-ADC was initially added to the cancer cells. Fig.18EShown are magnified photographs of T47D-wt cells treated with 1 μM paclitaxel for 72 hours. Fig.18F Shown are magnified photographs of T47D-MUC1* cells treated with 1 μM paclitaxel for 72 hours.

[0046] Fig.19 A graph showing cancer cell killing as a function of the concentration of 20A10-ADC MMAE added to the cell culture medium for 72 hours or 16 hours of treatment is shown. In the latter case, the medium was changed to medium without 20A10-ADC after 16 hours, and the experiment was allowed to continue until 72 hours after the first addition of 20A10-ADC to the cancer cells. Cell viability was measured using Presto Blue. Cell death was normalized to paclitaxel added to a final concentration of 1 μM, while the percentage of live cells at 72 hours was normalized to 0% viability.

[0047] Figures 20A-20B show fitted data graphs for measuring IC50s and data in tabular form. Figure 20A shows a fitted data graph of IC50 for cancer cell killing mediated by MNC2-ADC or 20A10-ADC, where the cancer cells are T47D-wt or T47D-MUC1*, and where the ADC is incubated with the target cancer cells for 16 hours or 72 hours. Figure 20B shows a table listing the IC50 of each ADC for each cell type and incubation condition.

[0048] Figure 21A-Figure 21C The MN20A10-OKT3 knob and hole form is shown. Fig.21A A photograph showing MUC1*-positive breast cancer cells T47D cultured with human T cells to which different concentrations of the bispecific antibody 20A10-OKT3-BiTE were added. 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 in the figure, the addition of the bispecific antibody mediates engagement of T cells and cancer cells, here manifested as T cell aggregation, a hallmark of activation directed by the bispecific bridge between T cell CD3 and cancer cell MUC1*. Fig. 21B Shown is a graph of secreted interferon-γ secreted by T cells as a function of added anti-MUC1* / anti-CD3 bispecific antibody, wherein the anti-MUC1* antibody is 20A10 and the anti-CD3 antibody is OKT3. Fig. 21C Shown is a graph of cell killing as a function of 20A10-OKT3 (anti-MUC1* / anti-CD3 bispecific antibody) concentration.

[0049] Figure 22A-22HVarious bispecific formats of MN20A10-OKT3 or MN20A10-12F6 are shown. The figure shows the killing curves of different formats of bispecific antibodies against T47D-wt or T47D-MUC1*. Fig. 22B Shown is a graph of T cell-mediated killing of cancer cells in the presence of the 12F6-MN20A10 bispecific. Fig.22A yes Fig. 22B Cartoon representation of the 12F6-MN20A10N bispecific format used in. Fig.22D Shown is a graph of T cell mediated killing of cancer cells in the presence of the MN20A10-12F6 KiH bispecific. Fig. 22C yes Fig.22D Cartoon representation of the KiH bispecific format used in. Fig.22F A graph showing T cell-mediated killing of cancer cells in the presence of the OKT3-MN20A10 bispecific is shown. Fig.22E yes Fig.22F Cartoon representation of the OKT3-MN20A10 bispecific format used in. Fig.22H Shown is a graph of T cell mediated killing of cancer cells in the presence of the MN20A10-12F6 chemically coupled bispecific. Figure 22G yes Fig.22H Cartoon representation of the MN20A10-12F6 bispecific format used in.

[0050] Fig.23 The IC50s of various bispecific formats of MN20A10-OKT3 or MN20A10-12F6 are shown.

[0051] Fig.24 The IC50s of various bispecific formats of MN20A10-OKT3 or MN20A10-12F6 are shown.

[0052] Figure 25A-25B A magnified photograph of human cancer cells stained with the anti-MUC1* antibody MNC2 is shown, where the cell nuclei are stained blue with DAPI and the MNC2 antibody fluoresces red. Fig.25A Shown is the attachment of the antibody to the cell surface at time zero. Fig.25B It shows that after 45 minutes, the antibody has been internalized and is visible throughout the cytoplasm. Antibody internalization is required for the ADC to function.

[0053] Fig.26The chemical structures of chemical entities including the toxic payload MMAE, which can be conjugated to an antibody to form an antibody-drug-conjugate, also known as an ADC, are shown. MC represents a maleimidocaproyl (MC) moiety that facilitates coupling to cysteine ​​on the antibody. VC represents a valine-citrulline (VC) moiety, and PAB represents a p-aminobenzyl (PAB) moiety, both of which facilitate enzymatic cleavage of the lysosomal enzyme cathepsin B within cancer cells. MMAE represents a monomethyl auristatin E (MMAE) moiety, which is a toxic payload that inhibits cell division by blocking tubulin polymerization, which is a toxic payload after cell internalization and / or cleavage of the non-toxic moiety.

[0054] Fig. 27 The chemical structures of chemical entities including the toxic payload MMAF are shown, which 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 coupling to cysteine ​​on the antibody. VALINE-CITRULLINE (VC) moiety and PAB represents p-aminobenzyl (PAB), both of which facilitate enzymatic cleavage of the toxic payload by the lysosomal enzyme cathepsin B in cancer cells. MMAF represents a monomethyl auristatin E (MMAF) moiety, which is a toxic payload after cell internalization and / or cleavage of the non-toxic moiety. The MMAF payload contains carboxylic acids, making it difficult (if not impossible) to expel the cell membrane after the payload is cleaved from the antibody. Therefore, MMAF can only show effective tubulin inhibition after the payload is internalized.

[0055] Fig.28 The chemical structure of the exatecan derivative Dxd is shown, which is incorporated into a reactive configuration called derutocan that is ready for conjugation to an antibody. MC represents a maleimidocaproyl (MC) moiety that facilitates coupling to a cysteine ​​on the antibody. GGFG represents a glycine-glycine-phenylalanine-glycine (GGFG) moiety that provides a flexible linker. Coupling agent represents a coupling agent (HN-CH2-O-CH2-CO) moiety that connects the linker and the payload via an ether diamide coupling agent. Exatecan represents the Dxd moiety that serves as a toxic payload after cellular internalization and / or cleavage of the non-toxic moiety.

[0056] Fig.29The chemical structure of exatecan derivatives is shown, which are incorporated into different chemical entities that facilitate conjugation to antibodies. MC represents the maleimidocaproyl (MC) moiety that facilitates coupling to cysteine ​​on the antibody. VC represents the valine-citrulline (VC) moiety, and PAB represents the p-aminobenzyl (PAB), both of which facilitate enzymatic cleavage of the toxic payload by the lysosomal enzyme cathepsin B within cancer cells. Exatecan represents the exatecan moiety, i.e., the toxic payload after cell internalization and / or cleavage of the non-toxic moiety.

[0057] Fig.30 A hydrophobic interaction chromatography (HIC) chromatogram of MNC2 conjugated to MMAE is shown. IgG1 antibodies, such as MNC2, have up to eight (8) cysteines with free thiols that can be conjugated to toxins or protoxins via the free thiols. Therefore, up to eight (8) toxins or payloads can be attached to each antibody. Analysis of the HIC chromatogram of MNC2 gave an average drug-to-antibody ratio (DAR) of 4.10.

[0058] Fig.31 The hydrophobic interaction chromatography (HIC) chromatogram of MNC2 coupled to MMAF is shown. The average DAR of MNC2-MMAF was determined to be 3.65.

[0059] Figure 32A-32B Shown are hydrophobic interaction chromatography (HIC) chromatograms of MNC2 conjugated to derutecan or exatecan. Fig.32A 32B shows the hydrophobic interaction chromatography (HIC) chromatogram of MNC2 conjugated to derutecan. The DAR of MNC2-derutecan was determined to be 7.7. Fig.32B The hydrophobic interaction chromatography (HIC) chromatogram of MNC2 conjugated to exatecan is shown. The DAR of MNC2-exatecan was determined to be 8.2.

[0060] Fig.33 The hydrophobic interaction chromatography (HIC) chromatogram of MN20A10 coupled with MMAE is shown. The average DAR of MN20A10-MMAE is 2.96.

[0061] Fig.34 The hydrophobic interaction chromatography (HIC) chromatogram of MN20A10 coupled to MMAF is shown. The average DAR of MN20A10-MMAF is 3.79.

[0062] Fig.35 The hydrophobic interaction chromatography (HIC) chromatogram of MN20A10 conjugated to delutecan is shown. The average DAR of MN20A10-delutecan is 4.6.

[0063] Figure 36A-Figure 36D Shown is a graph of flow cytometry measuring the ability of MNC2 to recognize cancer cells before and after conjugation to MMAE. Fig.36A The percentage of T47D breast cancer cells recognized by MNC2 before and after conjugation to MMAE is shown. Fig.36B The percentage of T47D breast cancer cells recognized by MNC2 after conjugation with MMAE is shown. Fig.36C The percentage of HCT-116, MUC1 negative cells recognized by MNC2 before or after conjugation to MMAE is shown. Fig.36D The percentage of NCI-H1975 lung cancer cells recognized by MNC2 after conjugation with MMAE is shown.

[0064] Figure 37A-Figure 37D Shown is a graph of flow cytometry measuring the ability of MN20A10 to recognize cancer cells before and after conjugation to MMAE. Fig.37A Shown are the percentages of T47D breast cancer cells recognized by MN20A10 before and after conjugation with MMAE. Fig.37B Shown are the percentages of T47D breast cancer cells recognized by MN20A10 after conjugation with MMAE. Fig.37C Shown is the percentage of HCT-116, MUC1 -negative cancer cells recognized by MN20A10 prior to conjugation with MMAE. Fig.37D Shown is the percentage of NCI-H1975 lung cancer cells recognized by MN20A10 after conjugation with MMAE.

[0065] Figure 38A-Figure 38F A graph of a plate reader assay is shown, where the viability of target cancer cells was measured as a function of the concentration of MNC2-ADC added, where viability was determined using PrestoBlue. The experiment was allowed to proceed for 72 hours before measurements were taken. Fig.38A Shown is the viability of T47D wild-type breast cancer cells after addition of MNC2-MMAE or MNC2-MMAF. Fig.38B Shown is the viability of T47D breast cancer cells engineered to express more MUC1* (referred to as T47D-MUC1*) after the addition of MNC2-MMAE or MNC2-MMAF. Fig.38C Shown is the viability of HPAF II wild-type pancreatic cancer cells after addition of MNC2-MMAE or MNC2-MMAF. Fig.38D Shown is the viability of HPAF II pancreatic cancer cells engineered to express more MUC1* (referred to as HPAF II-MUC1*) after the addition of MNC2-MMAE or MNC2-MMAF. Fig.38EShown is a table of calculated IC50s for the killing ability of MNC2-MMAE or MNC2-MMAF added to breast cancer cells or pancreatic cells expressing low levels of the target antigen MUC1*. Fig.38F Shown is a table of calculated IC50s for the killing ability of MNC2-MMAE or MNC2-MMAF added to breast cancer cells or pancreatic cells expressing high levels of the target antigen MUC1*.

[0066] Figure 39A-Figure 39F A graph of a plate reader assay is shown, where the viability of target cancer cells was measured as a function of the concentration of MN20A10-ADC added, where viability was determined using PrestoBlue. The experiment was allowed to proceed for 72 hours before measurements were taken. Fig.39A Shown is the viability of T47D wild-type breast cancer cells after addition of MN20A10-MMAE or MN20A10-MMAF. Fig.39B Shown is the viability of T47D breast cancer cells engineered to express more MUC1* (referred to as T47D-MUC1*) after addition of MN20A10-MMAE or MN20A10-MMAF. Fig.39C Shown is the viability of HPAF II wild-type pancreatic cancer cells after addition of MN20A10-MMAE or MN20A10-MMAF. Fig.39D Shown is the viability of HPAF II pancreatic cancer cells engineered to express more MUC1* (referred to as HPAF II-MUC1*) after addition of MN20A10-MMAE or MN20A10-MMAF. Fig.39E Shown is a table of calculated IC50s for the killing ability of MN20A10-MMAE or MN20A10-MMAF added to breast cancer cells or pancreatic cells expressing low to moderate levels of the target antigen MUC1*. Fig.39F Shown is a table of calculated IC50s for the killing ability of MN20A10-MMAE or MN20A10-MMAF added to breast cancer cells or pancreatic cells expressing high levels of the target antigen MUC1*.

[0067] Figure 40A-40C A graph of a plate reader assay is shown in which the viability of target cancer cells was measured as a function of the concentration of MNC2-ADC or MN20A10-ADC added, where viability was determined using PrestoBlue. Experiments with MMAE or derutocan conjugates were allowed to proceed for 72 or 120 hours, respectively, before measurements were taken. Fig.40A Shown is the viability of T47D wild-type breast cancer cells after addition of MNC2-MMAE, MNC2-deruteccan, MN20A10-MMAE, or MN20A10-deruteccan. Fig.40BShown is the viability of T47D breast cancer cells engineered to express more MUC1* (referred to as T47D-MUC1*) after addition of MNC2-MMAE, MNC2-drutecan, MN20A10-MMAE, or MN20A10-drutecan. Fig.40C Shown is a table of calculated IC50s for the killing ability of MNC2-MMAE, MNC2-derutecan, MN20A10-MMAE, or MN20A10-derutecan added to T47D breast cancer cells expressing medium to low MUC1* or high MUC1* T47D-MUC1* breast cancer cells.

[0068] Figure 41A-41B A graph of a plate reader assay is shown, in which the viability of target cancer cells was measured using PrestoBlue as a function of the concentration of MNC2-ADC or MN20A10-ADC added. The experiment was allowed to proceed for 72 hours before measurements were taken. Fig.41A Shown is the viability of DU145 wild-type hormone-resistant prostate cancer cells after addition of MNC2-MMAE or MN20A10-MMAE. Fig.41B The calculated IC50 table of the killing ability of MNC2-MMAE and MN20A10-MMAE against these prostate cancer cells is shown.

[0069] Figure 42A-42B A graph of a plate reader assay is shown, in which the viability of target cancer cells was measured using PrestoBlue as a function of the concentration of MNC2-ADC or MN20A10-ADC added. The experiment was allowed to proceed for 72 hours before measurements were taken. Fig.42A Shown is the viability of NCI-H1975 non-small cell lung cancer cells after addition of MNC2-MMAE or MN20A10-MMAE. Fig.42B The calculated IC50 table of the killing ability of MNC2-MMAE and MN20A10-MMAE on these lung cancer cells is shown.

[0070] Figure 43A-43C A graph of a plate reader assay is shown, in which the viability of target cancer cells was measured after the addition of MNC2-MMAE or MN20A10-MMAE. The experiment was allowed to proceed for 72 seconds before measurements were taken. Fig.43A Shown is the viability of T47D wild-type breast cancer cells after addition of MNC2-MMAE or MN20A10-MMAE. Fig.43B Shown is the viability of T47D breast cancer cells engineered to express more MUC1* (referred to as T47D-MUC1*) after addition of MNC2-MMAE or MN20A10-MMAE.

[0071] Figure 44A-Figure 44BA graph of a plate reader assay is shown comparing the viability of MUC1*-expressing cancer cells HCT-MUC1* and MUC1*-negative cancer cells HCT-116-wt after treatment with MN20A10-MMAE or IgG2b-MMAE (isotype control antibody). The experiment was allowed to proceed for 72 hours prior to flow cytometry measurements.

[0072] Figure 45A-Figure 45D Shown is a graph of a plate reader assay in which the viability of MUC1*-expressing cancer cells was measured following the addition of unconjugated MN20A10 or MN20A10-MMAE. Fig.45A The killing effect of MN20A10-MMAE on NCI H1975 non-small cell lung cancer cells is shown. Fig.45B The killing effect of MN20A10-MMAE on DU145 hormone-resistant prostate cancer cells is shown. Fig.45C Shown is the killing effect of MN20A10-MMAE on wild-type HPAF II pancreatic cancer cells or HPAF II-MUC1* cancer cells engineered to express more MUC1*. Fig.45D IC50 tables are shown.

[0073] Figure 46A-46J Magnified photographs of T47D-MUC1* breast cancer cells incubated with MNC2-MMAE or MNC2-drutecan are shown. Fig.46A The photograph taken at 4x magnification shows the killing effect of 500 nM MNC2-MMAE on breast cancer cells after 120 hours. Fig.46B The photograph taken at 4x magnification shows the killing effect of 6.2 nM MNC2-MMAE on breast cancer cells after 120 hours. Fig.46C Photographs taken at 4x magnification show untreated T47D breast cancer cells as a control. Fig.46D Shown is the killing effect of 500 nM MNC2-derutecan on breast cancer cells after 120 hours. Fig.46E The killing effect of 6.2 nM MNC2-derutecan on breast cancer cells after 120 hours was shown. Fig.46F Untreated T47D breast cancer cells are shown as a control. Figure 46G The photograph taken at 20x magnification shows the killing effect of 6.2 nM MNC2-MMAE on breast cancer cells after 120 hours. Figure 46H The photograph taken at 20x magnification shows untreated breast cancer cells as a control. Fig.46I The photograph taken at 20x magnification shows the killing effect of 6.2 nM MNC2-derutecan on breast cancer cells after 120 hours. Fig.46JThe photo taken at 20x magnification shows untreated breast cancer cells as a control. Killing can be easily seen as a significant reduction in cell number, changes in cell morphology to rounding and cell detachment. In contrast, the control wells show a confluent monolayer of dense cells with a flat spread morphology and no dead floating cells.

[0074] Figure 47A-47D Shown are photographs taken at 4x magnification of DU145 prostate cancer cells after incubation with MNC2-MMAE or MNC2-derutecan for 120 hours. Fig.47A The enlarged photograph shows the killing effect of MNC2-MMAE at 500 nM. Fig.47B Magnified photographs show normal morphology and confluence of untreated cells. Fig.47C The enlarged photograph shows the killing effect of MNC2-drutecan at 500 nM. Fig.47D Magnified photographs show untreated cells with normal morphology and confluence.

[0075] Figure 48A-48N Magnified photographs of T47D-MUC1* breast cancer cells incubated with MN20A10-MMAE or MN20A10-drutecan are shown. Fig.48A The photograph taken at 4x magnification shows the killing effect of 500 nM MN20A10-MMAE on breast cancer cells after 120 hours. Fig.48B The photograph taken at 4x magnification shows the killing effect of 56 nM MN20A10-MMAE on breast cancer cells after 120 hours. Fig.48C Photographs taken at 4x magnification show untreated T47D breast cancer cells as a control. Fig.48D Shown is the killing effect of 500 nM MN20A10-derutecan on breast cancer cells after 120 hours. Fig.48E The killing effect of 56 nM MN20A10-derutecan on breast cancer cells after 120 hours is shown. Fig.48F Untreated T47D breast cancer cells are shown as a control. Figure 48G The photograph taken at 20x magnification shows the killing effect of 56 nM MN20A10-MMAE on breast cancer cells after 120 hours. Figure 48H The photograph taken at 20x magnification shows untreated breast cancer cells as a control. Figure 48I The photograph taken at 20x magnification shows the killing effect of 56 nM MN20A10-derutecan on breast cancer cells after 120 hours. Fig.48J The photograph taken at 20x magnification shows untreated breast cancer cells as a control. Figure 48KPhoto taken at 20x magnification shows DU145 prostate cancer cells treated with 500 nM MN20A10-MMAE (DAR5.8). Treated cells show rounded and dead cells. Figure 48L Photographs taken at 20x magnification show untreated control DU145 prostate cancer cells. Figure 48M Photo taken at 20x magnification shows DU145 prostate cancer cells treated with 500 nM MN20A10-Drutecan (DAR 4.6). Treated cells show rounded and dead cells. Figure 48N Photographs taken at 20x magnification show untreated control DU145 prostate cancer cells.

[0076] Figure 49A-49J The trace of a real-time killing assay performed on an xCELLigence instrument is shown. In this assay, impedance is measured in real time. When adhered cancer cells are killed and fall off the electrode surface, the impedance (insulation) decreases. T47D wild-type breast cancer cells were plated onto multi-electrode well plates at 5,000 cells per well and allowed to adhere and grow for 24 hours before adding ADC. MNC2-MMAE (DAR 4.1), MN20A10-MMAE (DAR 5.8), MNC2-MMAF (DAR 3.7), MN20A10-MMAF (DAR 3.8), MNC2-Drutican (DAR 7.2), or MN20A10-Drutican (DAR 4.6) were then added to the cancer cells. Fig.49A The final concentration of MNC2-MMAE added is shown to be 500 nM, 167 nM or 2 nM and the experimental readout is 40 hours after ADC addition. Paclitaxel and Triton were added as positive killing controls. Fig.49B The final concentration of MN20A10-MMAE added is shown to be 167 nM, 56 nM or 2 nM and the experimental readout is 40 hours after ADC addition. Fig.49C The final concentration of MNC2-MMAF added is shown to be 500 nM, 167 nM or 2 nM and the experimental readout is 40 hours after ADC addition. Paclitaxel and Triton were added as positive killing controls. Fig.49D The final concentration of MN20A10-MMAF addition is shown to be 500 nM, 167 nM or 2 nM and the experimental readout is 40 hours after ADC addition. Fig.49E The final concentration of MNC2-MMAE addition is shown to be 500 nM, 167 nM or 2 nM, and the experimental readout is 120 hours after ADC addition. As shown, at a MNC2-MMAE concentration of 167 nM, essentially all tumor cells were killed. Fig.49FIt is shown that MNC2-MMAF was added at a final concentration of 500nM, 167nM or 2nM, and the experimental readout was 120 hours after ADC addition. As shown, at a MNC2-MMAF concentration of 167nM, essentially all tumor cells were killed. 49G shows that MNC2-Drutecan was added at a final concentration of 500nM, 167nM or 2nM, and the experimental readout was 120 hours after ADC addition. As shown, significant tumor cell killing occurred later than MNC2-MMAE or MNC2-MMAF, but at 100 hours, approximately the same level of killing was achieved at a MNC2-Drutecan concentration of 167nM. Figure 49H MN20A10-MMAE added over a range of concentrations is shown, and the experimental readout is 120 hours after ADC addition. As shown, at a MN20A10-MMAE concentration of 56 nM, essentially all tumor cells were killed. Fig.49I MN20A10-MMAF is shown added over a range of concentrations, and the experimental readout is 120 hours after ADC addition. As shown, at a MN20A10-MMAF concentration of 167 nM, essentially all tumor cells were killed. 49J shows MN20A10-Drutecan added over a range of concentrations, and the experimental readout is 120 hours after ADC addition. As shown, tumor cell killing occurs later than MN20A10-MMAE or MN20A10-MMAF, but at 120 hours, significant killing occurs at a MN20A10-Drutecan concentration of 167 nM.

[0077] Figures 50A-50J Shown is a trace of a real-time killing assay performed on an xCELLigence instrument. In this assay, impedance is measured in real time. Impedance (insulation) decreases as adherent cancer cells are killed and detached from the electrode surface. T47D-MUC1* breast cancer cells engineered to express more MUC1* were plated onto multi-electrode plates at 5,000 cells per well and allowed to adhere and grow for 24 hours before the ADC was added. MNC2-MMAE (DAR 4.1), MN20A10-MMAE (DAR 5.8), MNC2-MMAF (DAR 3.7), MN20A10-MMAF (DAR 3.8), MNC2-Drutecan (DAR 7.2), or MN20A10-Drutecan (DAR 4.6) were then added to the cancer cells. Fig.50A The final concentration of MNC2-MMAE added is shown to be 19nM, 6nM or 0.69nM, and the experimental readout is 40 hours after ADC addition. It can be seen that at 19nM, essentially all tumor cells were killed. Paclitaxel and Triton were added as positive killing controls. Fig.50B The final concentration of MN20A10-MMAE added is shown to be 19 nM, 6 nM or 2 nM, and the experimental readout is 40 hours after ADC addition. As can be seen, at 19 nM essentially all tumor cells were killed. Fig.50C The final concentration of MNC2-MMA added is shown to be 6 nM, 2 nM or 0.69 nM, and the experimental readout is 40 hours after ADC addition. It can be seen that at 6 nM, essentially all tumor cells were killed. Paclitaxel and Triton were added as positive killing controls. Fig.50D The final concentration of MN20A10-MMAF added is shown to be 56 nM, 19 nM or 2 nM, and the experimental readout is 40 hours after ADC addition. As can be seen, at 56 nM essentially all tumor cells were killed. Fig.50E MNC2-MMAE added over a range of concentrations is shown, and the experimental readout is 120 hours after ADC addition. As shown, at a MNC2-MMAE concentration of 6 nM, essentially all T47D-MUC1* tumor cells were killed. Fig.50F MNC2-MMAF is shown added over a range of concentrations, and the experimental readout is 120 hours after ADC addition. As shown, at a MNC2-MMAF concentration of 2 nM, essentially all T47D-MUC1* tumor cells were killed. 50G shows MNC2-Drutecan added over a range of concentrations, and the experimental readout is 120 hours after ADC addition. As shown, significant T47D-MUC1* tumor cell killing occurs later than MNC2-MMAE or MNC2-MMAF, but at 120 hours, approximately the same level of killing is achieved at a MNC2-Drutecan concentration of 6 nM. Figure 50H MN20A10-MMAE added over a range of concentrations is shown, and the experimental readout is 120 hours after ADC addition. As shown, at a MN20A10-MMAE concentration of 19 nM, essentially all tumor cells were killed. Fig.50I MN20A10-MMAF was added over a range of concentrations, and the experimental readout was 120 hours after ADC addition. As shown, at a MN20A10-MMAF concentration of 19 nM, essentially all tumor cells were killed. 50J shows MN20A10-Drutecan was added over a range of concentrations, and the experimental readout was 120 hours after ADC addition. As shown, T47D-MUC1* tumor cell killing occurred later than MN20A10-MMAE or MN20A10-MMAF, but at 120 hours, at a MN20A10-Drutecan concentration of 167 nM, essentially all tumor cells were killed.

[0078] Figure 51A-Figure 51I The trace of a real-time killing assay performed on an xCELLigence instrument is shown. In this assay, impedance is measured in real time. When adhered cancer cells are killed and fall off the electrode surface, the impedance (insulation) decreases. NCI-H1975 lung cancer cells were plated onto multi-electrode well plates at 5,000 cells per well and allowed to adhere and grow for 24 hours before adding ADC. MNC2-MMAE (DAR 4.1), MN20A10-MMAE (DAR 5.8), MNC2-MMAF (DAR 3.7), MN20A10-MMAF (DAR 3.8), MNC2-Drutican (DAR 7.2) or MN20A10-Drutican (DAR 4.6) were then added to the cancer cells. Fig.51A MNC2-MMAE added over a range of concentrations is shown, and the experimental readout is at 40 hours. As shown, essentially all lung tumor cells were killed at a concentration of 167 nM. Paclitaxel and Triton were added as positive killing controls. Fig.51B MN20A10-MMAE added over a range of concentrations is shown, and the experimental readout is at 40 hours. As shown, at a concentration of 167 nM, essentially all lung tumor cells were killed. Fig.51C MNC2-MMAF added over a range of concentrations is shown, and the experimental readout is at 40 hours. As shown, at a concentration of 167 nM, essentially all lung tumor cells were killed. Paclitaxel and Triton were added as positive killing controls. Fig.51D MN20A10-MMAF added over a range of concentrations is shown, and the experimental readout is at 40 hours. As shown, at a concentration of 167 nM, essentially all lung tumor cells were killed. Fig.51E MNC2-MMAE added over a range of concentrations is shown, and the experimental readout is 120 hours after ADC addition. As shown, at a MNC2-MMAE concentration of 167 nM, essentially all lung tumor cells were killed. Fig.51F MNC2-MMAF added over a range of concentrations is shown, and the experimental readout is 120 hours after ADC addition. As shown, at a MNC2-MMAF concentration of 56 nM, essentially all tumor cells were killed. 51G shows MNC2-Drutecan added over a range of concentrations, and the experimental readout is 120 hours after ADC addition. Fig.51H MN20A10-MMAE added over a range of concentrations is shown, and the experimental readout is 120 hours after ADC addition. As shown, at MN20A10-MMAE concentrations of 56 nM-19 nM, essentially all tumor cells were killed. Fig.51IMN20A10-MMAF added over a range of concentrations is shown, and the experimental readout is 120 hours after the addition of the ADC. As shown, at a MN20A10-MMAF concentration of 56 nM, essentially all tumor cells were killed.

[0079] Figure 52A-Figure 52I Shown is a trace of a real-time killing assay performed on an xCELLigence instrument. In this assay, impedance is measured in real time. When adhered cancer cells are killed and detached from the electrode surface, the impedance (insulation) decreases. HPAFII wild-type (WT) pancreatic cancer cells were plated onto a multi-electrode plate at 5,000 cells per well and allowed to adhere and grow for 24 hours before the ADC was added. MNC2-MMAE (DAR 4.1), MN20A10-MMAE (DAR 5.8), MNC2-MMAF (DAR 3.7), MN20A10-MMAF (DAR 3.8), MNC2-Drutecan (DAR 7.2), or MN20A10-Drutecan (DAR 4.6) were then added to the cancer cells. Fig.52A MNC2-MMAE added over a range of concentrations is shown, and the experimental readout was at 40 hours. Paclitaxel and Triton were added as positive killing controls. Fig.52B MN20A10-MMAE added over a range of concentrations is shown, and the experimental readout is at 40 hours. Fig.52C MNC2-MMAF added over a range of concentrations is shown, and the experimental readout was at 40 hours. Paclitaxel and Triton were added as positive killing controls. Fig.52D MN20A10-MMAF added over a range of concentrations is shown, and the experimental readout is at 40 hours. Fig.52E MNC2-MMAE added over a range of concentrations is shown, and the experimental readout is 120 hours after ADC addition. As shown, at a MNC2-MMAE concentration of 56 nM, essentially all pancreatic tumor cells were killed. Fig.52F MNC2-MMAF is shown added over a range of concentrations, and the experimental readout is 120 hours after ADC addition. As shown, at a MNC2-MMAF concentration of 56 nM, essentially all tumor cells were killed. 52G shows MNC2-Drutecan added over a range of concentrations, and the experimental readout is 120 hours after ADC addition. At 120 hours, significant killing was measured at a concentration of approximately 56 nM. Fig.52HMN20A10-MMAE added over a range of concentrations is shown, and the experimental readout is 120 hours after ADC addition. As shown, at MN20A10-MMAE concentrations of 56 nM-19 nM, essentially all tumor cells were killed. Fig.52I MN20A10-MMAF added over a range of concentrations is shown, and the experimental readout is 120 hours after ADC addition. As shown, at a MN20A10-MMAF concentration of 56 nM, essentially all tumor cells were killed.

[0080] Figure 53A-Figure 53I Shown is a trace of a real-time killing assay performed on an xCELLigence instrument. In this assay, impedance is measured in real time. Impedance (insulation) decreases as adherent cancer cells are killed and detached from the electrode surface. HPAF II-MUC1* pancreatic cancer cells engineered to express more MUC1* were plated onto multi-electrode well plates at 5,000 cells per well and allowed to adhere and grow for 24 hours before the ADC was added. MNC2-MMAE (DAR 4.1), MN20A10-MMAE (DAR 5.8), MNC2-MMAF (DAR 3.7), MN20A10-MMAF (DAR 3.8), MNC2-Drutecan (DAR 7.2), or MN20A10-Drutecan (DAR 4.6) were then added to the cancer cells. Fig.53A MNC2-MMAE added over a range of concentrations is shown, and the experimental readout was at 40 hours. Paclitaxel and Triton were added as positive killing controls. Fig.53B MN20A10-MMAE added over a range of concentrations is shown, and the experimental readout is at 40 hours. Fig.53C MNC2-MMAF added over a range of concentrations is shown, and the experimental readout was at 40 hours. Paclitaxel and Triton were added as positive killing controls. Fig.53D MN20A10-MMAF added at a range of concentrations is shown, and the experimental readout was at 40 hours. Fig.53E MNC2-MMAE added at a range of concentrations is shown, and the experimental readout is 120 hours after ADC addition. As shown, at a MNC2-MMAE concentration of 56 nM, essentially all pancreatic tumor cells were killed. Fig.53FMNC2-MMAF is shown added at a range of concentrations, and the experimental readout is 120 hours after ADC addition. As shown, at a MNC2-MMAF concentration of 56 nM, essentially all tumor cells were killed. 53G shows MNC2-Drutecan added at a range of concentrations, and the experimental readout is 120 hours after ADC addition. As can be seen from the figure, by 120 hours, at a concentration of 167 nM, almost all pancreatic cancer cells were killed. Fig.53H MN20A10-MMAE added over a range of concentrations is shown, and the experimental readout is 120 hours after ADC addition. As shown, at MN20A10-MMAE concentrations of 56 nM-19 nM, essentially all tumor cells were killed. Fig.53I MN20A10-MMAF added over a range of concentrations is shown, and the experimental readout is 120 hours after ADC addition. As shown, at a MN20A10-MMAF concentration of 56 nM, essentially all tumor cells were killed.

[0081] Figure 54A-Figure 54D Shown is a trace of a real-time killing assay performed on an xCELLigence instrument. In this assay, impedance is measured in real time. Impedance (insulation) decreases as adherent cancer cells are killed and detach from the electrode surface. Here, T47D-wt breast cancer cells expressing low to moderate levels of MUC1* were plated onto a multi-electrode 96-well plate at 5,000 cells per well and allowed to adhere and grow for 24 hours before the ADC was added. MNC2-exatecan (DAR 8.2), MNC2-drutecan (DAR 4.0), MNC2-MMAE (DAR 4.1), or 50 μM of free exatecan were then added to the cancer cells. The xCELLigence readout was 0 to 120 hours. Fig.54A Various ADCs added at a concentration of 500 nM are shown. Fig.54B Various ADCs added at a concentration of 167 nM are shown. Fig.54C Various ADCs added at a concentration of 56 nM are shown. Fig.54D Various ADCs added at a concentration of 19 nM are shown. Figure 54E shows a legend listing the color of each ADC track and the DAR of each ADC. As can be seen from the figure, MNC2-exatecan effectively kills target cancer cells at concentrations as low as 56 nM.

[0082] Figure 55A-Figure 55DShown is a trace of a real-time killing assay performed on an xCELLigence instrument. In this assay, impedance is measured in real time. Impedance (insulation) decreases as adherent cancer cells are killed and detached from the electrode surface. Here, T47D-MUC1* breast cancer cells engineered to express high levels of MUC1* were plated onto a multi-electrode 96-well plate at 5,000 cells per well and allowed to adhere and grow for 24 hours before the ADC was added. MNC2-exatecan (DAR 8.2), MNC2-drutecan (DAR 4.0), MNC2-MMAE (DAR 4.1), or 50 μM of free exatecan were then added to the cancer cells. The xCELLigence readout was from 0 to 120 hours. Fig.55A Various ADCs added at a concentration of 19 nM are shown. Fig.55B Various ADCs added at a concentration of 6.2 nM are shown. Fig.55C Various ADCs added at a concentration of 2.1 nM are shown. Fig.55D Various ADCs added at a concentration of 0.69 nM are shown. Figure 55E shows a legend listing the color of each ADC track and the DAR of each ADC. As can be seen from the figure, MNC2-exatecan and MNC2-drutecan effectively kill cancer cells expressing high levels of target antigen at concentrations as low as 6.2 nM and even as low as 2.1 nM.

[0083] Figure 56A-Figure 56D Shown is a trace of a real-time killing assay performed on an xCELLigence instrument. In this assay, impedance is measured in real time. As adherent cancer cells are killed and detached from the electrode surface, the impedance (insulation) decreases. Here, NCI-H1975 non-small cell lung cancer cells expressing very low levels of MUC1* were plated onto a multi-electrode 96-well plate at 5,000 cells per well and allowed to adhere and grow for 24 hours before the ADC was added. MNC2-exatecan (DAR 8.2), MNC2-derutecan (DAR 4.0), MNC2-MMAE (DAR 4.1), or 50 μM of free exatecan were then added to the cancer cells. The xCELLigence readout was from 0 to 120 hours. Fig.56A Various ADCs added at a concentration of 500 nM are shown. Fig.56B Various ADCs added at a concentration of 167 nM are shown. Fig.56C Various ADCs added at a concentration of 56 nM are shown. Fig.56D Various ADCs added at a concentration of 19 nM are shown. Figure 56E shows a legend listing the color of each ADC track and the DAR of each ADC. As can be seen from the figure, MNC2-exatecan effectively kills cancer cells expressing very low levels of target antigen at concentrations as low as 167 nM.

[0084] Figure 57A-Figure 57F Bioluminescent images are shown 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*. Animals were injected with 5 mg / kg of MNC2-MMAE (DAR 3.9) on day 7 after tumor implantation, but injections were increased to 10 mg / kg on days 14 and 20. Fig.57A Control animals implanted with T47D-wt tumor cells but mock-injected with PBS are shown. Fig.57B Shown are animals implanted with T47D-wt cells and then injected with MNC2-MMAE. Fig.57C Shown are control animals implanted with T47D-MUCl* tumor cells but mock injected with PBS. Fig.57D Shown are animals implanted with T47D-MUC1* cells and then injected with MNC2-MMAE. Fig.57E The bioluminescence (radiance photons / cm 2 ) as a function of days post tumor implantation in mice implanted with T47D wild-type breast cancer cells. Fig.57F The bioluminescence (radiance photons / cm 2 ) as a function of days post tumor implantation in mice implanted with T47D-MUC1* breast cancer cells.

[0085] Figure 58A-Figure 58F Bioluminescent images are shown 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*. Animals were injected with 5 mg / kg of MN20A10-MMAE (DAR 3.0) on day 7 after tumor implantation, but injections were increased to 10 mg / kg on days 14 and 20. Fig.58A Control animals implanted with T47D-wt tumor cells but mock-injected with PBS are shown. Fig.58B Shown are animals implanted with T47D-wt cells and then injected with MN20A10-MMAE. Fig.58C Shown are control animals implanted with T47D-MUCl* tumor cells but mock injected with PBS. Fig.58DShown are animals implanted with T47D-MUC1* cells and then injected with MN20A10-MMAE. Fig.58E The bioluminescence (radiance photons / cm 2 ) as a function of days post tumor implantation in mice implanted with T47D wild-type breast cancer cells. Fig.58F Bioluminescence (radiance photons / sec / cm 2 ) as a function of days post tumor implantation in mice implanted with T47D-MUC1* breast cancer cells.

[0086] Figure 59A-Figure 59C Bioluminescent images of female nu / nu mice implanted in the right flank with 1M human NCI-H1975 non-small cell lung cancer cells expressing low to moderate levels of MUC1* are shown. On day 7 after tumor implantation, animals were injected with 5mg / kg or 10mg / kg of MNC2-MMAE as indicated. Increasing the dose of MNC2-MMAE (DAR 3.9) to 10mg / kg increased tumor cell killing on days 19 and 27 as can be seen in the bioluminescent images. Fig.59A Shown are control animals implanted with NCI-H1975 non-small cell lung cancer cells but mock injected with PBS. Fig.59B Shown are animals implanted with NCI-H1975 non-small cell lung cancer cells and then injected with MNC2-MMAE. Fig.59C Bioluminescence (radiance photons / sec / cm 2 ) as a function of days after tumor implantation.

[0087] Figure 60A-60D Bioluminescent photographs taken on an IVIS instrument of female nu / nu mice implanted in the right flank with 1M human NCI-H1975 non-small cell lung cancer cells expressing low to moderate levels of MUC1* are shown. Animals were injected with 5 mg / kg of MN20A10-MMAE (DAR 3.0) on days 7 and 14 after tumor implantation. On day 19, the dose was increased to 10 mg / kg and administered by intraperitoneal (ip) injection. As can be seen from the photographs and graphs, the increased dose significantly increased the killing of tumor cells. Fig.60A Shown are control animals implanted with NCI-H1975 non-small cell lung cancer cells but mock injected with PBS. Fig.60B Shown are animals implanted with NCI-H1975 non-small cell lung cancer cells and then injected with MN20A10-MMAE. Fig.60C Photographs and weights of tumors excised from control animals on day 26 are shown. Fig.60DBioluminescence (radiance photons / sec / cm 2 ) as a function of days after tumor implantation.

[0088] Figure 61A-61V Bioluminescent images taken on an IVIS instrument are shown of female nu / nu mice implanted in the right flank with 0.5M human pancreatic cancer cells, either HPAFII wild-type (wt) cells expressing low to moderate levels of MUC1* or HPAF II-MUC1* cells engineered to express more MUC1*. On day 7 after tumor implantation, animals were injected with either PBS as a control or 10 mg / kg of MNC2-MMAE (DAR 4.1). Fig.61A Shown are IVIS bioluminescent images of control animals implanted with HPAF II-wt pancreatic cancer cells but mock-injected with PBS. Fig.61B Shown are IVIS bioluminescence photographs of animals implanted with HPAF II-wt pancreatic cancer cells and then injected with MNC2-MMAE. Fig.61C Shown are IVIS bioluminescent images of control animals implanted with HPAF II-MUC1* pancreatic cancer cells but mock injected with PBS. Fig.61D Shown are IVIS bioluminescence photographs of animals implanted with HPAF II-MUCl* pancreatic cancer cells and then injected with MNC2-MMAE. Fig.61E Shown are photographs of tumors excised from HPAF II-wt control animals that had to be sacrificed due to excessive tumor burden. Fig.61F Shown are photographs of tumors resected from HPAF II-MUCl* control animals that had to be sacrificed due to excessive tumor burden. Figure 61G Bioluminescence (radiance photons / sec / cm2) of both control and MNC2-MMAE treated mice are shown. 2 ) as a function of days after tumor implantation. The day 25 IVIS data point was omitted due to instrument failure. Fig.61H Shown is a bar graph of caliper measurements of tumors at day 25. Caliper measurements eliminate differences in luciferase expression levels in the HPAFII-wt cell line and the HPAF II-MUC1* cell line. Fig.61I Shown are day 11 photographs of control animals implanted with HPAFII-wt pancreatic cancer cells but mock-injected with PBS. Fig.61J Shown are day 18 photographs of control animals implanted with HPAF II-wt pancreatic cancer cells but mock-injected with PBS. Figure 61KShown are day 25 photographs of control animals implanted with HPAF II-wt pancreatic cancer cells but mock-injected with PBS, showing an increasing tumor size in the right flank. Figure 61L Shown are day 11 photographs of animals implanted with HPAF II-wt pancreatic cancer cells followed by injection of MNC2-MMAE. Figure 61M Shown are day 18 photographs of animals implanted with HPAF II-wt pancreatic cancer cells followed by injection of MNC2-MMAE. Figure 61N Shown are day 25 photographs of animals implanted with HPAFII-wt pancreatic cancer cells and then injected with MNC2-MMAE, with 3 mice having small, barely visible tumors. Fig.61O Shown are day 11 photographs of control animals implanted with HPAF II-MUC1* pancreatic cancer cells but mock injected with PBS. Figure 61P Shown are day 18 photographs of control animals implanted with HPAF II-MUC1* pancreatic cancer cells but mock injected with PBS. Figure 61Q Shown is a day 25 photograph of a control animal implanted with HPAF II-MUC1* pancreatic cancer cells but mock injected with PBS, showing a large tumor in the right flank. Figure 61R Shown are day 11 photographs of animals implanted with HPAF II-MUCl* pancreatic cancer cells and then injected with MNC2-MMAE. Figure 61S Shown are day 18 photographs of animals implanted with HPAF II-MUCl* pancreatic cancer cells and then injected with MNC2-MMAE. Figure 61T Shown is a day 25 photograph of an animal implanted with HPAF II-MUCl* pancreatic cancer cells and then injected with MNC2-MMAE, in which no tumors could be seen or felt.

[0089] Figure 62A-62F Shown are bioluminescent photographs taken on an IVIS instrument of female nu / nu mice that were implanted in the right flank with 0.5M human pancreatic cancer HPAF II-MUC1* cells engineered to express more MUC1*. On day 7 after tumor implantation, the animals were injected with PBS as a control or 10mg / kg of MNC2-MMAE (DAR4.1). As can be seen in the figure, by day 66, the treated mice had no tumors or residual tumor spots. Since the HPAF II cell line does not express luciferase well, the bioluminescence is weak. For this reason, we also performed caliper measurements, which record the actual size of the tumor. Fig.62A Shown are IVIS bioluminescent images of control animals implanted with HPAF II-MUC1* pancreatic cancer cells but mock injected with PBS. Fig.61BShown are IVIS bioluminescent images of animals implanted with HPAF II-MUCl* pancreatic cancer cells and then injected with 10 mg / kg of MNC2-MMAE. Fig.62C Shown are photographs of tumors excised from HPAF II-MUCl* control animals that had to be sacrificed due to excessive tumor burden, compared to only spots of tumor or no tumor left in the MNC2-MMAE treated groups. Fig.62D Bioluminescence (radiance photons / sec / cm2) of both control and MNC2-MMAE treated mice are shown. 2 ) as a function of days after tumor implantation. Fig.62E Kaplan-Meier survival plots of control and treated mice are shown. Fig.62F Bar graphs showing caliper measurements for control and treated animals are shown.

[0090] Figure 63A-63H Bioluminescent images 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*. On day 6 after tumor implantation, animals were injected with 10 mg / kg of MNC2-derutecan (DAR 4.2), but this was increased to 20 mg / kg for T47D-wt treated mice. Treatment of T47D-MUC1* treated mice was kept constant at 10 mg / kg. Fig.63A Control animals implanted with T47D-wt tumor cells but mock-injected with PBS are shown. Fig.63B Shown are animals implanted with T47D-wt cells and then injected with MNC2-delutec. Fig.63C Shown are control animals implanted with T47D-MUCl* tumor cells but mock injected with PBS. Fig.63D Shown are animals implanted with T47D-MUC1* cells and then injected with MNC2-delutec. Fig.63E The bioluminescence (radiance photons / cm 2 ) as a function of days post tumor implantation in mice implanted with T47D wild-type breast cancer cells. Fig.63F Shown is a bar graph of bioluminescence measurements per mouse per day for animals implanted with T47D-wt breast cancer cells. Figure 63G Bioluminescence (radiance photons / sec / cm 2 ) as a function of days post tumor implantation in mice implanted with T47D-MUC1* breast cancer cells. Figure 63H Shown is a bar graph of bioluminescence measurements per mouse per day for animals implanted with T47D-MUC1* breast cancer cells.

[0091] Figure 64A-Figure 64T Line graphs of IVIS luminescence measurements for individual mice from day 4 to day 30 are shown. NOD / SCOD / GAMMA mice were implanted with either T47D-wt breast cancer cells or T47D-MUC1* cells that express more MUC1*. Both groups of mice were mock treated with PBS or treated with MNC2-Drutecan. On day 6 after tumor implantation, animals were injected with MNC2-Drutecan (DAR 4.2) at 10 mg / kg, but this was increased to 20 mg / kg for T47D-wt treated mice. Treatment of T47D-MUC1* treated mice remained constant at 10 mg / kg. Fig.64A Shown is the growth of T47D-wt tumor in mouse #1 from the control group. Fig.64B Shown is the growth of T47D-wt tumor in mouse #2 from the control group. Fig.64C Shown is the growth of T47D-wt tumor in mouse #3 from the control group. Fig.64D Shown is the growth of T47D-wt tumor in mouse #4 from the control group. Figure 64E Shown is the growth of T47D-wt tumor in mouse #5 from the control group. Fig.64F Shown is the growth of T47D-wt tumor in mouse #1 of the group treated with MNC2-delutec. Figure 64G Shown is the growth of T47D-wt tumor in mouse #2 of the group treated with MNC2-delutec. Figure 64H Shown is the growth of T47D-wt tumor in mouse #3 of the group treated with MNC2-delutec. Figure 64I Shown is the growth of T47D-wt tumor in mouse #4 of the group treated with MNC2-delutec. Figure 64J Shown is the growth of T47D-wt tumor in mouse #5 of the group treated with MNC2-delutec. Figure 64K Shown is the growth of a T47D-MUCl* tumor in mouse #1 in the control group. Figure 64L Shown is the growth of a T47D-MUCl* tumor in mouse #2 in the control group. Figure 64M Shown is the growth of a T47D-MUCl* tumor in mouse #3 in the control group. Figure 64N Shown is the growth of a T47D-MUCl* tumor in mouse #4 in the control group. Fig.64O Shown is the growth of a T47D-MUCl* tumor in mouse #5 in the control group. Figure 64P Shown is the growth of T47D-MUC1* tumor in mouse #1 of the group treated with MNC2-derutecan. Figure 64Q Shown is the growth of T47D-MUCl* tumor in mouse #2 of the group treated with MNC2-derutecan. Figure 64R Shown is the growth of T47D-MUCl* tumor in mouse #3 of the group treated with MNC2-delutec. Figure 64S Shown is the growth of T47D-MUCl* tumor in mouse #4 of the group treated with MNC2-derutecan. Figure 64T Shown is the growth of T47D-MUCl* tumor in mouse #5 of the group treated with MNC2-derutecan.

[0092] Figure 65A-Figure 65D Images of tumor cells are shown. Staining of day 0 cells shows that the cells express more full-length MUC1 than MUC1*, and the staining intensity is light, indicating a low number of MUC1* receptors, which indicates an early stage cancer. 62 days after tumor implantation, which is equivalent to about 7 years in human time, it can be easily seen that MUC1* expression (in terms of expression level and expression intensity) has significantly changed from low expression to high expression in advanced tumors. In contrast, staining of serial sections of the tumor shows expression of full-length MUC1 as expected, because it is cleaved into MUC1* after surface expression. However, the staining intensity did not increase, indicating that the vast majority of MUC1 expressed in advanced tumors has been cleaved into the growth factor receptor form MUC1*. DETAILED DESCRIPTION

[0093] The present application relates to anti-MUC1* multispecific antibodies and anti-MUC1* antibody conjugates and methods of making and using the same. The cleaved form of the MUC1 transmembrane protein is a growth factor receptor that drives the growth of more than 75% of all human cancers. The cleaved form of MUC1, MUC1* (pronounced muk 1 star), is a growth factor receptor. Cleavage and release of a large portion of the MUC1 extracellular domain reveals a binding site for activating ligand dimerization NME1, NME6, NME7, NME7AB, NME7-X1 or NME8. It is an ideal target for cancer drugs because it is aberrantly expressed in more than 75% of all cancers and is likely overexpressed in an even higher proportion of metastatic cancers (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 adeterminant of trastuzumab (Herceptin) resistance in breast cancer cells," Breast Cancer Res Treat. 118(1):113-124). After MUC1 is cleaved, most of its extracellular domain falls off the cell surface. The remaining part of MUC1* has a truncated extracellular domain that contains most or all of the major growth factor receptor sequence PSMGFR (SEQ ID NO:133).

[0094] Many drugs currently administered to patients parenterally are not targeted, resulting in systemic delivery of the drug to cells and tissues of the body, which is unnecessary and often undesirable. This can lead to adverse drug side effects and often limits the dose of drugs (e.g., chemotherapeutic (anti-cancer), cytotoxic, enzyme inhibitor agents, and antiviral or antibacterial drugs) that can be administered.

[0095] A major goal is to develop methods for specifically targeting therapeutic agents to cells and tissues. The benefits of such treatments include avoiding the general physiological effects that would otherwise result from inappropriate delivery of such agents to other cells and tissues. The disclosure herein addresses these and other limitations and problems of the past.

[0096] Specific terms

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those skilled in the art of the claimed subject matter generally understood. Unless otherwise indicated, all patents, patent applications, published applications and publications, GENBANK sequences, websites and other published materials mentioned in the entire disclosure herein are incorporated by reference in their entirety. Usually, the rules of cell culture, cell infection, antibody production and molecular biology methods are methods commonly used in this area. This standard technique can be found in, for example, reference manuals (such as Sambrook et al. (2000) and Ausubel et al. (1994)).

[0098] 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 otherwise clearly stated. As used herein, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" as well as other forms such as "include", "includes", and "included" is not limiting.

[0099] 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 in the claim. The transitional phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0100] As used herein, ranges and amounts may be expressed as "about" a particular value or range. About also includes an exact amount. For example, "about 1 mg" means "about 1 mg" as well as "1 mg." The terms "about" and "approximately" generally include amounts that are expected to be within the range of experimental error.

[0101] The terms "individual," "patient," or "subject" are used interchangeably. As used herein, they refer to any mammal (i.e., species of any order, family, and genus within the animal kingdom: chordates: vertebrates: mammalia). In some embodiments, the mammal is a human. None of these terms require or are limited to situations characterized by supervision (e.g., continuous or intermittent) by a health care worker (e.g., a doctor, registered nurse, licensed nurse, physician assistant, caregiver, or hospice worker).

[0102] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs). These terms encompass 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.

[0103] When amino acid sequences are provided herein, L-amino acid, D-amino acid or β amino acid versions of the sequence and reverse, inverted and reverse inverted isomers are also considered. Peptide also includes amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids, and naturally occurring amino acid polymers. In addition, the term is applicable to amino acids connected by peptide bonds or other modified bonds (for example, wherein peptide bonds are replaced by α-esters, β-esters, thioamides, phosphoamides, carbamates, hydroxylates, etc. (see, for example, Spatola, (1983) Chem.Biochem.Amino Acids and Proteins 7: 267-357), wherein amides are replaced by saturated amines (see, for example, Skiles et al., U.S. Patent number 4,496,542, which is incorporated herein by reference, and Kaltenbronn et al., (1990) 969-970 pages, Proc.11th American Peptide Symposium, ESCOM Science Publishers, The Netherlands, etc.)).

[0104] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acids are divided into hydrophobic amino acids, polar amino acids, nonpolar amino acids, 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, aspartic acid, glutamic acid, and analogs thereof. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as 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 natural amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of amino acids, but function similarly to natural amino acids. Amino acids are either D amino acids or L amino acids.

[0105] As used in the specification and appended claims, unless otherwise indicated, the following terms have the meanings set forth below.

[0106] In some embodiments, the compounds disclosed herein contain one or more asymmetric centers, thus producing enantiomers, diastereomers, and other stereoisomeric forms, which are defined as (R) or (S) in terms of absolute stereochemistry. Unless otherwise stated, the present disclosure is intended to cover all stereoisomeric forms of the compounds disclosed herein. When the compounds described herein contain olefin double bonds, unless otherwise stated, the present disclosure is intended to include E and Z geometric isomers (e.g., cis or trans). Therefore, the compounds provided herein can be enantiomerically pure or can be stereoisomers or diastereomeric mixtures. The compounds provided herein can contain chiral centers. Such chiral centers can be (R) or (S) configurations, or mixtures thereof. The chiral centers of the compounds provided herein can undergo epimerization in vivo. Therefore, those skilled in the art will recognize that for compounds that undergo epimerization in vivo, administering the compound in the (R) form is equivalent to administering the compound in the (S) form. Similarly, all possible isomers and their racemic and optically pure forms, as well as all tautomeric forms, are also intended to be included. The term "geometric isomer" refers to E or Z geometric isomers (eg, cis or trans) of an olefin 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.

[0107] "Tautomers" refers to molecules in which a proton can be transferred from one atom of a molecule to another atom of the same molecule. In certain embodiments, the compounds presented herein exist as tautomers. Where tautomerism is possible, a chemical equilibrium of the tautomers will exist. The exact ratio of the tautomers depends on several factors, including physical state, temperature, solvent, and pH. Some examples of tautomeric equilibrium include:

[0108]

[0109] "Pharmaceutically acceptable salts" include both acid addition salts and base addition salts. Pharmaceutically acceptable salts of any of the compounds or conjugates described herein are intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0110] As used herein, the terms "antibody" and "immunoglobulin" are technical terms and are used interchangeably herein to refer to molecules having an antigen binding site that specifically binds to an antigen. In certain embodiments, the isolated antibody (e.g., monoclonal antibody) or antigen-binding fragment thereof described herein specifically binds to a target protein.

[0111] Antibodies can include, for example, 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 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., identical pairs), intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, bivalent antibodies (including monospecific or bispecific bivalent antibodies), single chain antibodies or single chain variable fragments (scFv), camelized antibodies, affibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and epitope-binding fragments of any of the above.

[0112] The antibody can be 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 molecules. In certain embodiments, the antibodies described herein are IgG antibodies (e.g., human IgG), or their classes (e.g., human IgG1, IgG2, IgG3 or IgG4) or subclasses.

[0113] The CDR sequences of the antibodies disclosed herein or the anti-MUC1* or anti-CD3 binding domain sequences disclosed herein can be defined or determined according to: (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 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242); or (ii) the Chothia numbering scheme, which is referred to herein as the "Chothia CDR" (see, 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., 1990, J. Mol. Biol. 215(1):175-82; and U.S. Pat. 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 also, 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).

[0114] With regard to the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which may optionally include one or two additional amino acids, following 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). It is well known to those skilled in the art that, using the Kabat numbering system, the actual linear amino acid sequence of an antibody variable domain may contain fewer or more amino acids due to shortening or lengthening of FRs and / or CDRs, and therefore the Kabat numbering of an amino acid is not necessarily the same as its linear amino acid numbering.

[0115] 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.

[0116] The term "multispecific" refers to the ability of an antibody to specifically bind to two or more different antigenic determinants, e.g., two binding sites, each formed by a pair of antibody heavy chain variable domain (VH) and antibody light chain variable domain (VL) that bind to different antigens.

[0117] The term "human antibody" or "humanized antibody" as used herein 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, transgenic animals (e.g., mice) also produce human antibodies, which are able to produce a full set or selected human antibodies in the absence of endogenous immunoglobulin production after immunization. Transferring the human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., J 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 other cases, human antibodies are also 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. can also be used to prepare 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).

[0118] As used herein, an "antigen" is a moiety or molecule that contains an epitope to which an antibody can specifically bind. Thus, an antigen is specifically bound by an antibody. In certain embodiments, the antigen bound by an antibody described herein is a target protein, such as MUC1*, CD3, or a fragment thereof.

[0119] As used herein, the term "heavy chain" when applied to antibodies may refer to any of the different types based on the amino acid sequence of the constant domains, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and muon (μ), which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including the IgG subclasses, such as IgG1, IgG2, IgG3, and IgG4.

[0120] As used herein, the term "light chain" when used for antibodies can refer to any of the different types based on the constant domain amino acid sequence, such as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0121] As used herein, the term "amino acid sequence identity percentage (%)" or "sequence identity" of a relevant sequence is defined as the percentage of the amino acid residue in the candidate sequence identical with the amino acid residue in the particular sequence after comparing sequences and introducing room (if necessary) to realize maximum sequence identity percentage, and not considering any conservative substitution as a part for sequence identity. The comparison for determining amino acid sequence identity percentage can be realized by the various means within the technical scope 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 the comparison, including any algorithm required for maximum comparison on the total length of the compared sequence.

[0122] In the case of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity between a given amino acid sequence A and a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity with a given amino acid sequence B) is calculated as follows: 100 multiplied by a fraction X / Y, where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of A and B, and Y is the total number of amino acid residues in B. It is understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity between A and B will not be equal to the % amino acid sequence identity between B and A. Unless expressly stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.

[0123] The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than antibody fragments as defined below. These terms specifically refer to antibodies having heavy chains that include an Fc region.

[0124] "Antibody fragments" comprise only a portion of an intact antibody, wherein the portion retains at least one, two, three, and up to most or all of the functions normally associated with the portion in the intact antibody. In one aspect, an antibody fragment comprises the antigen binding site of the intact antibody, and thus retains the ability to bind to an antigen.

[0125] As used herein, "epitope" is a term known in the art and refers to a local region of an antigen to which an antibody can specifically bind. An epitope can be a linear epitope of continuous amino acids or can include amino acids from two or more discontinuous regions of an antigen.

[0126] As used herein, the terms "bind," "binds to," "specifically binds," or "specifically binds to" in the context of antibody binding refer to the binding of an antibody to an antigen (e.g., an epitope), as binding is understood by those skilled in the art. In certain embodiments, the affinity (K) of a molecule that specifically binds to an antigen is the affinity (K) of the molecule that specifically binds to the antigen. d ) is at least greater than the K of the molecule when it binds to another antigen. d 2 logs, 2.5 logs, 3 logs, 4 logs lower (higher affinity). In another specific embodiment, the molecule that specifically binds to an antigen does not cross-react with other proteins.

[0127] A "linking moiety" or "linker" (e.g., denoted L) is a molecule with two reactive ends, one for conjugation to a polypeptide (e.g., antibody) via a conjugating moiety Y, and the other for conjugation to a linking moiety (denoted SP) or, when SP is absent, to a moiety T. The polypeptide conjugation reactive end of the linker is typically a site capable of conjugation to a polypeptide (e.g., antibody) via a cysteine ​​thiol group on the polypeptide (e.g., antibody), and is therefore typically a thiol-reactive group, such as maleimide or dibromomaleimide, or as defined herein.

[0128] As used herein, "treatment" or "treating" or "alleviation" or "improvement" are used interchangeably. These terms refer to methods of obtaining beneficial or desired results (including but not limited to therapeutic benefit and / or prophylactic benefit). "Therapeutic benefit" refers to the eradication or improvement of the underlying condition being treated. In addition, therapeutic benefit is achieved by eradication or improvement of one or more physiological symptoms associated with the underlying condition, such that improvement is observed in the patient, even though the patient still suffers from the underlying condition. For prophylactic benefit, in some embodiments, the composition is administered to a patient at risk for a particular disease, or to a patient reporting one or more physiological symptoms of a disease, even if a diagnosis of the disease has not yet been made.

[0129] MUC1* binding domain

[0130] 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 its antigen-binding fragment or variant is a monoclonal antibody. In some embodiments, the antibody or its antigen-binding fragment or variant is a human antibody, a mouse 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.

[0131] In some embodiments, the antibody or functional fragment or functional variant thereof that specifically binds to MUCl* comprises an anti-MUCl* heavy chain and an anti-MUCl* light chain.

[0132] 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 a 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 a variable domain of a kappa or lambda light chain.

[0133] 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 κ or λ 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 κ or λ 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 κ or λ 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 κ or λ light chain.

[0134] 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.

[0135] 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.

[0136] In some embodiments, the antibody or its functional fragment or functional variant that specifically binds to MUC1* comprises a single chain variable fragment (scFv) or an antigen binding fragment (Fab). In some embodiments, the antibody or its functional fragment or functional variant that specifically binds to MUC1* comprises a single chain variable fragment. In some embodiments, the antibody or its functional fragment or functional variant that specifically binds to MUC1* comprises an antigen binding fragment (Fab).

[0137] In some embodiments, the anti-MUC1* heavy chain variable domain includes complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the anti-MUC1* heavy chain variable domain include an amino acid sequence according to: HC-CDR1: SEQ ID NO: 1 or 4; HC-CDR2: SEQ ID NO: 2 or 5; HC-CDR3: SEQ ID NO: 3 or 6; and wherein the CDRs comprise 0-2 amino acid modifications (e.g., 0 or 1 amino acid modification) in at least one of HC-CDR1, HC-CDR2, or HC-CDR3.

[0138] In some embodiments, the anti-MUC1* light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the anti-MUC1* light chain variable domain comprise an amino acid sequence according to: LC-CDR1: SEQ ID NO: 13 or 16; LC-CDR2: SEQ ID NO: 14 or 17; LC-CDR3: SEQ ID NO: 15 or 18; and wherein the CDRs comprise 0-2 amino acid modifications (e.g., 0 or 1 amino acid modifications) in at least one of LC-CDR1, LC-CDR2, or LC-CDR3.

[0139] In some embodiments, the anti-MUCl* heavy chain comprises an amino acid sequence 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% identical to SEQ ID NO:38 or 44. In some embodiments, the anti-MUCl* light chain comprises an amino acid sequence 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% identical to SEQ ID NO:41 or 47.

[0140] Table 1. MUC1* binding domain CDR sequences

[0141]

[0142] Table 2: Examples of heavy chain sequences, heavy chain variable domain sequences, light chain sequences, and light chain variable domain sequences of anti-MUC1* binding domains

[0143]

[0144]

[0145]

[0146]

[0147] Table 3: Anti-MUC1* single chain variable fragment (scFv) sequences

[0148]

[0149] CD3 binding domain

[0150] 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 its antigen binding fragment or variant is a monoclonal antibody. In some embodiments, the antibody or its antigen binding fragment or variant is a human antibody, a mouse 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.

[0151] 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.

[0152] In some embodiments, the antibody that specifically binds to CD3 or a functional fragment or functional variant thereof comprises an anti-CD3 heavy chain and an anti-CD3 light chain.

[0153] 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 the 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 the variable domain of a κ or λ light chain. In some embodiments, the anti-CD3 heavy chain variable domain comprises the variable domain of an IgG1 heavy chain, and the anti-CD3 light chain variable domain comprises the variable domain of a κ or λ light chain. In some embodiments, the anti-CD3 heavy chain variable domain comprises the variable domain of an IgG2 heavy chain, and the anti-CD3 light chain variable domain comprises the variable domain of a κ or λ light chain. In some embodiments, the anti-CD3 heavy chain variable domain comprises the variable domain of an IgG3 heavy chain, and the anti-CD3 light chain variable domain comprises the variable domain of a κ or λ light chain. In some embodiments, the anti-CD3 heavy chain variable domain comprises the variable domain of an IgG4 heavy chain, and the anti-CD3 light chain variable domain comprises the variable domain of a kappa or lambda light chain.

[0154] In some embodiments, the antibody or its functional fragment or functional variant that specifically binds to CD3 comprises a single chain variable fragment (scFv) or an antigen binding fragment (Fab). In some embodiments, the antibody or its functional fragment or functional variant that specifically binds to CD3 comprises a single chain variable fragment (scFv). In some embodiments, the antibody or its functional fragment or functional variant that specifically binds to CD3 comprises an antigen binding fragment (Fab).

[0155] In some embodiments, the anti-CD3 heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the anti-CD3 heavy chain variable domain comprise an amino acid sequence according to: HC-CDR1: SEQ ID NO: 7 or 10; HC-CDR2: SEQ ID NO: 8 or 11; HC-CDR3: SEQ ID NO: 9 or 12; and wherein the CDRs comprise 0-2 amino acid modifications (e.g., 0 or 1 amino acid modification) in at least one of HC-CDR1, HC-CDR2, or HC-CDR3.

[0156] In some embodiments, the anti-CD3 light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the anti-CD3 light chain variable domain comprise an amino acid sequence according to: LC-CDR1: SEQ ID NO: 19 or 22; LC-CDR2: SEQ ID NO: 20 or 23; LC-CDR3: SEQ ID NO: 21 or 24; and wherein the CDR comprises 0-2 amino acid modifications (e.g., 0 or 1 amino acid modification) in at least one of LC-CDR1, LC-CDR2, or LC-CDR3.

[0157] In some embodiments, the anti-CD3 heavy chain comprises an amino acid sequence that is 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% identical to SEQ ID NO:26 or 32.

[0158] ] In some embodiments, the anti-CD3 heavy chain comprises an amino acid sequence that is 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% identical to SEQ ID NO:30 or 33.

[0159] In some embodiments, the anti-CD3 heavy chain comprises an amino acid sequence that is 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% identical to any of the amino acid sequences in Table 5.

[0160] In some embodiments, the anti-CD3 light chain comprises an amino acid sequence that is 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% identical to any of the amino acid sequences in Table 5.

[0161] Table 4: Anti-CD3 CDRs

[0162]

[0163] Table 5: Examples of heavy chain sequences, heavy chain variable domain sequences, light chain sequences, and light chain variable domain sequences of anti-CD3 binding domains

[0164]

[0165]

[0166]

[0167]

[0168] Table 6: Anti-CD3 single chain variable fragment (scFv) sequences

[0169]

[0170] Antibodies that bind to MUC1* and CD3

[0171] In some embodiments, the antibody or a functional fragment or a functional variant thereof that specifically binds to MUCl* comprises a Fab, and the antibody or a functional fragment or a functional variant thereof that specifically binds to CD3 comprises a scFv.

[0172] In some embodiments, the anti-MUCl* heavy chain comprises an amino acid sequence 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% identical to SEQ ID NO:38 or 44, and the anti-MUCl* light chain comprises an amino acid sequence 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% identical to SEQ ID NO:41 or 47; and the scFv comprises an amino acid sequence 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% identical to SEQ ID NO: NO:131 or 132 has an amino acid sequence that is 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% identical.

[0173] In some embodiments, the antibody or a functional fragment or a functional variant thereof that specifically binds to MUCl* comprises a scFv, and the antibody or a functional fragment or a functional variant thereof that specifically binds to CD3 comprises a Fab.

[0174] In some embodiments, the anti-CD3 heavy chain comprises an amino acid sequence that is 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% identical to SEQ ID NO:29, 30, 35, or 36; and the scFv comprises an amino acid sequence that is 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% identical to SEQ ID NO:29, 30, 35, or 36; ID NO: 129 or 130 has an amino acid sequence that is 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% identical.

[0175] In some embodiments, the antibody comprises at least 3 CDRs of an anti-MUC1* binding domain selected from any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 13, 14, 15, 16, 17 or 18 and 0-2 amino acid modifications (e.g., 0-1 amino acid modifications) thereof; and at least 3 CDRs of an anti-CD3 binding domain selected from any one of SEQ ID NO: 7, 8, 9, 10, 11, 12, 19, 20, 21, 22, 23 or 24 and 0-2 amino acid modifications (e.g., 0-1 amino acid modifications) thereof.

[0176] In some embodiments, the antibody or a functional fragment or a functional variant thereof that specifically binds to MUCl* comprises a scFv, and the antibody or a functional fragment or a functional variant thereof that binds to CD3 comprises a scFv.

[0177] In some embodiments, the anti-MUCl* scFv comprises an amino acid sequence 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% identical to SEQ ID NO: 129 or 130. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence 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% identical to SEQ ID NO: 131 or 132.

[0178] Table 7: Exemplary sequences of antibodies that bind to MUC1* and CD3

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220] In some embodiments, the antibody further comprises a crystallizable fragment (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 (e.g., human) IgG1, IgG2, IgG3, or IgG4 domains. In some embodiments, the heterodimeric Fc region comprises (e.g., human) IgG1 domains. In some embodiments, the heterodimeric Fc region comprises (e.g., human) IgG2 domains. In some embodiments, the heterodimeric Fc region comprises (e.g., human) IgG3 domains. In some embodiments, the heterodimeric Fc region comprises (e.g., human) IgG4 domains.

[0221] In some embodiments, the heterodimeric Fc region, wherein the heterodimeric Fc region comprises a knob chain and a hole chain, forms a knob-hole (KIH) structure (Spiess et al. Molecular Immunology 67, 95-106 (2015)). 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 mortar chain comprises a (eg, human) IgG4 domain.

[0222] In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer is breast cancer, colon cancer, prostate cancer, pancreatic cancer, or lung cancer.

[0223] In some embodiments, the antibody binds to cancer cells that express MUCl* on their surface.

[0224] As an example of how the antibodies of the present invention can be incorporated into a bispecific antibody, a bispecific antibody was constructed using the knob-in-hole (also known as KIH) (Spiess et al. Molecular Immunology 67, 95-106 (2015)) format. 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; the second arm of the antibody is 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 herein as 20A10-OKT3-BiTE and 20A10-12F6-BiTE. In a functional demonstration, the bispecific antibodies were added at various concentrations to cultured cells in which human T cells and MUC1*-positive cancer cells were present. In one case, the cancer cells were T47D breast cancer cells, and in another case, the MUC1*-negative line HCT-116 colon cancer cells had been transduced to express MUC1*, referred to as HCT-MUC1*. From Figure 1A-1L , Figure 2A-2L , Figure 3A-Figure 3L and Figure 4A-4L As can be seen in the photos shown, the addition of either bispecific antibody mediated engagement of T cells and MUC1*-positive cancer cells, as evidenced by bispecific dose-dependent cell aggregation. Two control experiments were performed. In one control, no bispecific antibody was added. In the other control, only MUC1* cancer cells were present. No aggregation was observed. In another control, the bispecific antibody was added to the MUC1*-positive cancer cells, but no T cells were present. In addition, cancer cell killing was quantified. The cytotoxicity of the anti-MUC1*-targeted bispecific antibodies was measured using the LDH cytotoxicity assay, in which 20A10 binds to the MUC1* extracellular domain on the surface of tumor cells and an anti-CD3 antibody called OKT3. A cartoon depicting how the LDH cytotoxicity assay works is shown ( Figure 1M ), where higher values ​​of A490 indicate greater cell killing. Figure 1NA graph of cell killing as a function of 20A10-OKT3 (anti-MUC1* / anti-CD3 bispecific antibody) concentration is shown. As can be seen from 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, is a potent killer of MUC1* positive tumor cells. In addition to analyzing target cancer cells, T cells were also analyzed. It is well known that activated CD8-positive cytotoxic T cells secrete interferon-gamma (IFN-g) when they are activated and ready to kill. Secretion of IFN-g ( Fig.1O ). As can be seen from the figure, IFN-g reaches half-maximal secretion at a low bispecific concentration of 12.3 ng / mL. Activated T cells also secrete TNF-α into the conditioned medium. ELISA measurement of TNF-a also shows half-maximal secretion at a very low concentration of 12.3 ng / mL of the 20A10-CD3 bispecific antibody ( Figure 1P ).

[0225] In another example, 20A10 was used to construct an anti-MUC1* / anti-CD3 bispecific antibody for binding to the extracellular domain of MUC1*-positive cancer cells, and an anti-CD3 antibody 12F6 for binding to T cells. Figure 2A-2L As can be seen in the photos shown in , the addition of either bispecific antibody mediated engagement of T cells and MUC1*-positive cancer cells, as evidenced by bispecific dose-dependent cell aggregation. Two control experiments were performed. In one control, no bispecific antibody was added. In the other control, only MUC1* cancer cells were present. No aggregation was observed. In another control, the bispecific antibody was added to the MUC1*-positive cancer cells, but no T cells were present. In addition, cancer cell killing was quantified. The cytotoxicity of the anti-MUC1*-targeted bispecific antibodies was measured using the LDH cytotoxicity assay, in which 20A10 binds to the MUC1* extracellular domain on the surface of tumor cells and an anti-CD3 antibody called 12F6. A cartoon depicting the working principle of the LDH cytotoxicity assay is shown ( Figure 2M ), where higher values ​​of A490 indicate greater cell killing. Figure 2NA graph of cell killing as a function of 20A10-12F6 (anti-MUC1* / anti-CD3 bispecific antibody) concentration is shown. As can be seen from 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, is a potent killer of MUC1* positive tumor cells. In addition to analyzing target cancer cells, T cells were also analyzed. It is well known that activated CD8-positive cytotoxic T cells secrete interferon-gamma (IFN-g) when they are activated and ready to kill. Secretion of IFN-g ( Fig.2O ). As can be seen from the figure, IFN-g reaches half-maximal secretion at a low bispecific concentration of 50 ng / mL. Activated T cells also secrete TNF-α into the conditioned medium. ELISA measurement of TNF-a also shows half-maximal secretion at a very low concentration of 200 ng / mL of the 20A10-CD3 bispecific antibody ( Figure 2P The difference in efficacy of 20A10 paired with OKT3 compared to 12F6 suggests that the efficacy of anti-MUC1* / anti-T cell is modulated by the respective affinity and specificity of the two antibodies.

[0226] In another example, the anti-MUC1* antibody 20A10 was paired with an anti-CD3 antibody fragment and tested for killing of MUC1* negative line HCT-116 colon cancer cells that had been transduced to express MUC1*, referred to as HCT-MUC1*. In one case, the antibody fragment used to bind to T cells was OKT3. In another case, the anti-CD3 antibody was 12F6. Figure 3A-Figure 3L (OKT3) and Figure 4A-4L As can be seen in the photographs of (12F6), the addition of either bispecific antibody mediated engagement of T cells and MUC1* positive cancer cells, as evidenced by bispecific dose-dependent cell aggregation. Two control experiments were performed. In one control, no bispecific antibody was added, but both T cells and MUC1* cancer cells were present. No aggregation was observed. In the other control, the bispecific antibody was added to MUC1* positive cancer cells, but no T cells were present.

[0227] Antibody-drug conjugates

[0228] Antibody-drug conjugates (ADCs) allow for targeted delivery of therapeutic agents to specific cells and / or tissues. In certain embodiments, the ADC includes an agent (e.g., a therapeutic agent) that can inhibit a topoisomerase (e.g., topoisomerase I (TopoI)) or inhibit tubulin production.

[0229] In another aspect, conjugates are provided herein, e.g., antibody-drug conjugates. The ADC comprises a therapeutic agent (denoted as X); a linker (denoted as L); a coupling moiety (denoted as R); and a cysteine ​​thiol group that binds to the antibody (denoted as ) of the conjugated moiety (denoted as Z). In some embodiments, intracellular cleavage of the L linker allows the therapeutic agent X to be removed from the separation, thereby promoting the uptake or retention of therapeutic agent X into cells or tissues.

[0230] In certain embodiments, the ADC comprises 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 coupling moiety; L is a dipeptide, tripeptide or tetrapeptide linking moiety having Z bound to the N-terminus and R bound 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 MUC1*HC-CDR3 comprises an amino acid sequence selected from those specified in Table 1; wherein 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, MUC1*LC-CDR2 and MUC1*LC-CDR3 of the MUC1* binding domain comprise an amino acid sequence selected from those specified in Table 1; Z is a conjugated moiety capable of forming a covalent bond with the sulfur atom of a cysteine ​​residue; and wherein y is an integer from 1 to 10.

[0231] The ADC may include the structure provided below, where n is 1 to 10: The ADC may include the structure provided below, where n is 1 to 10:

[0232] The efficacy of an ADC is determined in part by the drug-to-antibody ratio, or DAR. Basically, the more toxin attached to your antibody, the more cell killing there is. However, attaching too much toxin to the antibody can destabilize the antibody or sterically hinder 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 an antibody-drug-conjugate (ADC). HIC columns on a high-pressure liquid chromatography (HPLC) system are used to analyze and characterize ADCs using salt gradient buffers. HIC separates proteins based on differences in their surface hydrophobicity, and the more drug bound to the antibody, the longer the retention time. Figure 30-35 HIC chromatograms of one batch of MNC2 and one batch of MN20A10 conjugated with several toxic payloads are shown along with the corresponding calculated DARs for each.

[0233] In certain embodiments, the conjugates provided herein have a DAR ranging from 1 to 20. In certain embodiments, the conjugates provided herein have a DAR ranging from 1 to 15. In certain embodiments, the conjugates provided herein have a DAR ranging from 1 to 10. In certain embodiments, the conjugates provided herein have a DAR ranging from 1 to 8. In certain embodiments, the conjugates provided herein have a DAR ranging from 1 to 7. In certain embodiments, the conjugates provided herein have a DAR ranging from 1 to 6. In certain embodiments, the conjugates provided herein have a DAR ranging from 1 to 5. In certain embodiments, the conjugates provided herein have a DAR ranging from 1 to 4.

[0234] 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.

[0235] 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.

[0236] In certain embodiments, fewer than the theoretical maximum number of units are conjugated to a polypeptide (eg, an antibody) during a conjugation reaction.

[0237] In certain embodiments, the amino acid attached to the unit is located in the heavy chain of the antibody. In certain embodiments, the amino acid attached to the unit is located in the light chain of the antibody. In certain embodiments, the amino acid attached to the unit is located in the hinge region of the antibody. In certain embodiments, the amino acid attached to the unit is located in the Fc region of the antibody. In certain embodiments, the amino acid attached to the unit is located in the constant region (e.g., CH1, CH2 or CH3 of the heavy chain, or CH1 of the light chain) of the antibody. In other embodiments, the amino acid attached to the unit or the drug unit is located in the VH framework region of the antibody. In other embodiments, the amino acid attached to the unit is located in the VL framework region of the antibody.

[0238] It should be understood that the preparation of the conjugates described herein can produce a mixture of conjugates with one or more units attached to a polypeptide (i.e., heterogeneous) such as an antibody. Individual conjugate molecules can be identified in the mixture by mass spectrometry and separated by HPLC (e.g., hydrophobic interaction chromatography, including such methods known in the art). In certain embodiments, a homogeneous conjugate with a single DAR (loading) value can be separated from a conjugated mixture by electrophoresis or chromatography.

[0239] The present disclosure provides an antibody-drug conjugate (ADC) comprising a monoclonal antibody or an antigen-binding fragment thereof directed against MUC1* conjugated to a cytotoxin. The term "antibody-drug conjugate" as used herein refers to a compound comprising a monoclonal antibody (mAb) connected to a cytotoxic agent (usually a small molecule drug with high systemic toxicity) via a chemical linker. In some embodiments, the ADC may comprise a small molecule cytotoxin that has been chemically modified to comprise a linker. The cytotoxin is then conjugated to the antibody or its antigen-binding fragment using a linker. After binding to a target antigen on the cell surface, the ADC is internalized and transported to a lysosome, where the cytotoxin is released by proteolysis of a cleavable linker (e.g., by cathepsin B in the lysosome) or by proteolytic degradation of the antibody (if connected to the cytotoxin via a non-cleavable linker). The cytotoxin is then transported from the lysosome to the cytoplasm or nucleus, where it can then bind to its target according to its mechanism of action.

[0240] The antibody-drug conjugates described herein may include whole antibodies or antibody fragments. The parent antibody may be mouse, rabbit, human, humanized, camel or other species.

[0241] ADC can include an antigen-binding fragment of an antibody. 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 can 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, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of two domains of the Fv fragment (i.e., VL and VH) connected by a synthetic linker that enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242:424). 3-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16:778 (1998)) and (v) diabodies, which are dimers of polypeptide chains, each of which comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between VH and VL on the same polypeptide chain, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to generate a dimeric molecule with two functional antigen-binding sites.

[0242] The terms "cytotoxin" and "cytotoxic agent" refer to any molecule that inhibits or prevents cell function and / or causes cell destruction (cell death) and / or exerts an anti-proliferative effect. It should be understood that the cytotoxin or cytotoxic agent of an ADC is also referred to as the "payload" of the ADC in the art. Many classes of cytotoxic agents are known in the art to have potential utility in ADC molecules and can be used for the ADCs described herein. These classes of cytotoxic agents include, for example, anti-microtubule agents (e.g., auristatins and maytansinoids), pyrrolobenzodiazepines, (PBD), RNA polymerase II inhibitors (e.g., amatoxin), and DNA alkylating agents (e.g., indolebenzodiazepines Examples of specific cytotoxic agents that can be used in the ADCs described herein include, but are not limited to, amanitin, auristatins, calicheamicin, daunorubicin, doxorubicin, duocarmycins, dolastatins, enediynes, lexitropsins, taxanes, puromycin, maytansine alkaloids, vinca alkaloids, tubulysin, and pyrrolobenzodiazepines. More specifically, the cytotoxic agent can be, for example, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholinodoxorubicin, rhizosin, cyanomorpholinodoxorubicin, dolastatin-10, echinomycin, combretastatin, chalicheamicin, maytansine, DM1, DM4, vinblastine, methotrexate, fusobacterium, or a derivative or analog thereof.

[0243] Auristatins represent a class of highly potent antimitotic agents that have demonstrated significant preclinical activity at well-tolerated doses. Examples of auristatins that can be used in combination with the ADCs described herein include, but are not limited to, monomethyl auristatin E (MMAE) and the related molecule monomethyl auristatin F (MMAF).

[0244] In one embodiment, the cytotoxic agent may be a pyrrolobenzodiazepine (PBD) or PBD derivatives. PBD is transported into the cell nucleus, where it crosslinks DNA, prevents replication during mitosis, damages DNA by inducing single-strand breaks, and subsequently causes apoptosis. Some PBDs also have the ability to recognize and bind to specific DNA sequences.

[0245] The anti-MUC1* monoclonal antibodies described herein comprise at least one cytotoxic molecule conjugated thereto; however, the anti-MUC1* monoclonal antibodies may comprise any suitable number of cytotoxic molecules conjugated thereto (e.g., 1, 2, 3, 4, or more cytotoxic molecules) to achieve the desired therapeutic effect.

[0246] The present disclosure also provides compositions comprising the above-mentioned antibodies or antibody-drug conjugates and pharmaceutically acceptable (e.g., physiologically acceptable) carriers. Any suitable carrier known in the art can be used within the scope of the present invention. The choice of carrier will be determined in part by the specific site to which the composition can be applied and the specific method for applying the composition. The composition can optionally be sterile. The composition can be generated according to the conventional techniques described in, for example, Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2001).

[0247] The composition preferably comprises an amount of the antibody or antibody-drug conjugate effective to treat or prevent a cancer expressing MUC1*. Thus, the present 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.

[0248] The present disclosure also provides use of an antibody or ADC described herein, or a composition comprising an antibody or ADC, in the manufacture of a medicament for treating a MUC1*-positive cancer.

[0249] As shown herein, MUC1* is expressed on a variety of cancer types. Therefore, the present disclosure provides a method of killing such cancer cells, comprising contacting a cancer cell expressing MUC1* with an antibody-drug conjugate described herein or a composition comprising an ADC described herein, whereby the antibody-drug conjugate binds to MUC1* on the cell and kills the cell.

[0250] The antibody-drug conjugates described herein or compositions comprising the antibody-drug conjugates can be contacted with a cell population expressing MUCl* ex vivo, in vivo, or in vitro (preferably in vivo).

[0251] 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 cures the disease and / or the adverse symptoms caused by the disease. To this end, the methods of the present invention include administering a "therapeutically effective amount" of an antibody or ADC or a composition comprising an antibody or ADC and a pharmaceutically acceptable carrier. A "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic result at a dosage and for a necessary period of time. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex and 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.

[0252] Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., the effect 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 an ADC to a mammal susceptible to a cancer expressing MUC1*. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic outcome (e.g., preventing the onset of disease).

[0253] The efficacy of treatment or prevention can be monitored by periodically assessing the patient receiving treatment. In one embodiment, the ADC described herein inhibits or suppresses the proliferation of cells expressing MUC1* 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 integration 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)).

[0254] The antibodies or ADCs described herein or compositions comprising antibodies or ADCs can be administered to mammals (e.g., humans) using standard administration techniques (including, e.g., intravenous, intraperitoneal, subcutaneous). More preferably, the antibodies or ADCs or compositions comprising antibodies or ADCs are administered to mammals by intravenous injection.

[0255] The antibodies or ADCs described herein or compositions comprising antibodies or ADCs can be administered together with one or more additional therapeutic agents, which can be co-administered to a mammal. The term "co-administered" as used herein refers to the administration of one or more additional therapeutic agents and antibodies or ADCs described herein or compositions containing antibodies or ADCs, close enough in time so that the antibodies or ADCs can enhance the effect of one or more additional therapeutic agents, or vice versa. In this regard, antibodies or ADCs or compositions containing them can be administered first, and one or more additional therapeutic agents can be administered secondly, or vice versa. For example, antibodies or ADCs or compositions containing them can be administered in combination with other agents (e.g., as adjuvants) to treat or prevent MUC1*-positive cancers. In this regard, antibodies or ADCs or compositions containing antibodies or ADCs can be used in combination with at least one other anticancer agent, and at least one other anticancer agent includes, for example, any suitable chemotherapeutic agent known in the art, ionizing radiation, small molecule anticancer agents, cancer vaccines, biological therapies (e.g., other monoclonal antibodies, cancer killer viruses, gene therapy, and adoptive T cell transfer) and / or surgery.

[0256] As an example of how the antibodies of the present invention can be incorporated into ADCs, antibody drug conjugates, to treat MUC1* positive cancers, a toxin is attached to the anti-MUC1* antibody MNC2. In this particular example, the toxin is MMAE, monomethyl auristatin, although a variety of toxins and functional linkers known to those skilled in the art can promote ADC-directed killing of target cells, especially cancer cells. In this example, MNC2 is deglycosylated and then reacted with a linker that promotes covalent attachment of MMAE, referred to herein as MNC2-ADC. Different concentrations of MNC2-ADC are incubated with target cells for different time periods. The cells are then photographed and labeled using a PCR product called PrestoBlue TM Cell viability was measured using a cell death indicator (Thermo Fisher Scientific, Waltham, MA). MNC2-ADC incubated with HCT (which is a MUC1 and MUC1* negative cell line) did not induce cell death at any concentration ( Figure 5A-5F ). However, when HCT cells were transduced to express MUC1*, generating HCT-MUC1*, MNC2-ADC did induce cell death in a dose-dependent manner ( Figure 6A-6G ). Fig. 9B A graph showing the measured loss of cell viability is shown. Similarly, MNC2-ADC did not induce cell death in K562 cells, which are MUC1* negative cells ( Figure 7A-7G ). Similarly, when cells were transduced to express MUC1*, generating K562-MUC1*, MNC2-ADC was able to induce cell death in a concentration-dependent manner ( Figure 8A-8G ). Fig.9A Graphs showing the measured loss of cell viability are shown.

[0257] T47D cells are a naturally occurring breast cancer cell line that express full-length MUC1 (to which MNC2 does not bind) as well as MUC1* (to which MNC2 binds). T47D-MUC1* is a cell line in which T47D cells have been transduced to express even more MUC1*. Figures 10A-10G ) or T47D-MUC1* cells ( Figures 11A-11G ) were incubated together. From the chart of measuring cell viability ( Fig.12 ) that only T47D-MUC1* cells were killed at the highest concentration tested here. Another experiment was performed in which a much higher concentration of MNC2-ADC was added. In this case, T47D-WT cells were killed by MNC2-ADC at 1000 nM ( Figure 13A-13J ), and T47D-MUC1* cells were killed at 10 nM, 39 nM, 100 nM, 393 nM and at 1000 nM ( Figure 14A-14J ). Fig.15A Graphs showing measurement of T47D and T47D-MUC1* cell death induced by MNC2-ADC are shown. These experiments are shown as an example only. In order for the ADC to work, engagement of the ADC with the target cell must induce receptor internalization, which is demonstrated by this set of examples. The toxin attached to the anti-MUC1* antibody can be one of several toxins known to those skilled in the art. Similarly, MNC2 can be human or humanized.

[0258] However, optimization of the toxin conjugation chemistry greatly improved the results of MNC2-ADC. In the next experiment, a valine-citrulline p-aminobenzylcarbamate (VC-PAB) linker was used to connect the deglycosylated antibody and toxin. One end of the linker was conjugated to MMAE alone, and then the other end was reacted with maleimide and then conjugated to the antibody after reduction by alkylation. MMAE inhibits cell division by blocking the polymerization of tubulin. The stable linker outside the cell is cleaved by cathepsin B and the ADC is internalized, thereby activating MMAE. This optimized MNC2-ADC concentration ranged from 0.1ng / mL to 500ng / mL, compared with T47D wild type (-WT) ( Fig.16A and Fig. 16C ) or T47D cells transduced to express more MUC1* (T47D-MUC1*) ( Fig. 16B and Fig.16D In one case, MNC2-ADC was incubated with 5,000 target MUC1*-positive cells per well of a 96-well plate for 72 hours ( Figure 16A-16B Alternatively, MNC2-ADC was incubated with target MUC1*-positive cells only for 16 h, after which the medium was replaced and the cells continued to be cultured for an additional 54 h ( Figure 16C-16D For comparison, the well-known cancer drug paclitaxel was added to T47D-wt or T47D-MUC1* cells for 72 hours ( Figure 16E-16F ). Using a method called PrestoBlue TM The viability of the 72-h co-cultures was measured with a cell death indicator (Thermo Fisher Scientific, Waltham, MA). Fig.17 ). As can be seen from the figure, cells with higher antigen density were killed faster and at lower MNC2-ADC concentrations. In the 72-hour incubation, 90% of high MUC1* cells were killed at an MNC2-ADC concentration of 100 nM.

[0259] 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 the remaining cancer cells were taken after 72 hours of co-culture with 20A10-ADC ( Figure 18A-18F ) In this particular example, the toxin is MMAE, monomethyl auristatin, although a variety of toxins and functional linkers are known to those skilled in the art to promote ADC-directed killing of target cells (especially cancer cells). 20A10 was deglycosylated and then reacted with a linker that promotes covalent attachment of MMAE, referred to herein as 20A10-ADC or 20A10-MMAE. 20A10-ADC at concentrations ranging from 0.1 ng / mL to 500 ng / mL was co-administered with T47D wild-type (-WT) ( Fig.18A and Fig.18C ) or T47D cells transduced to express more MUC1* (T47D-MUC1*) ( Fig.18B and Fig.18D ). In one case, 20A10-ADC was incubated with target MUC1*-positive cells for the entire 72-hour assay ( Figure 18A-18B Alternatively, 20A10-ADC was incubated with target MUC1*-positive cells only for 16 h, after which the medium was replaced and the cells continued to be cultured for an additional 54 h ( Figure 18C-Figure 18D For comparison, the well-known cancer drug paclitaxel was added to T47D-wt or T47D-MUC1* cells for 72 hours ( Figure 18E-18F ). Using a method called PrestoBlue TMThe viability of the 72-h co-cultures was measured using a cell death indicator (Thermo Fisher Scientific, Waltham, MA). Fig.19 ). As can be seen from the graph, the higher the antigen density, the more cancer cells are killed. Even after 16 hours of incubation, more than 80% of the high MUC1* cells were killed, and after 72 hours of incubation, 100% were killed at concentrations below 10 nM. Cells expressing lower amounts of MUC1* require higher 20A10-ADC concentrations and longer incubation time periods to achieve 100% cell killing.

[0260] The IC50s for these experiments are plotted in the graph of Figure 20A. The data are also presented in tabular form in Figure 20B.

[0261] These experiments demonstrate that anti-MUC1* antibodies can be successfully incorporated into a number of ADC formats using optimized conjugation chemistries as well as new and improved toxins and linkers.

[0262] One of the in vitro methods to characterize each ADC is to measure the ability of the antibody to bind the target antigen before and after the toxin is conjugated to the antibody. The specificity of the antibody can be compromised by the process of chemically conjugating multiple toxins to the antibody. This phenomenon is more due to the intrinsic stability or instability of each antibody than to elements of the conjugation process. In many cases, the payload is conjugated to the antibody by binding to free thiols, which are generated by breaking or reducing disulfide bonds. Disulfide bonds hold the two heavy chains together and support the structure of the variable region, which is the antibody recognition unit. A significant challenge is to reduce enough disulfide bonds to allow attachment of multiple toxins without disrupting the disulfide bonds that maintain critical antibody structure. Surprisingly, MNC2 is a very stable, well-behaved antibody that allows attachment of multiple toxins without disrupting the antibody's structure or changing its ability to recognize the target antigen. Challenges encountered when attaching payloads to the antibody MN20A10 involve the timing of disulfide bond reduction and toxin conjugation. Most ADC conjugation protocols require iterative disulfide bond reduction followed by testing the number of free thiols. If this is a prolonged process, the free thiol can be reoxidized, which can re-establish the original structure or can generate a new structure that results in loss of antibody target specificity. This occurred when attempting to conjugate the toxin to the antibody MN20A10, which is an IgG2b isotype. This problem was overcome by 1) empirically determining the number of molar equivalents required to reduce MN20A10; 2) adding this number of reducing equivalents all at once; 3) adding the coupling reagent at an elevated temperature, where the time to reach the required temperature can be reduced by performing the reaction in a water bath, and further speeding up the reaction time by intermittent shaking during the reaction. Fig.36A – Fig.36DShown is a graph of flow cytometry measurements of the ability of MNC2 to recognize breast cancer cells and lung cancer cells before and after conjugation to MMAE. Fig.37A – Fig.37D Graphs showing flow cytometry measurements of the ability of MN20A10 to recognize breast and lung cancer cells before and after conjugation to MMAE are shown. MNC2 appears to be less able to recognize cancer cells after conjugation to MMAE than MN20A10. Given that the drug-to-antibody ratio (DAR) of MNC2-MMAE was higher than that of 20A10-MMAE in this experiment, coupled with the greater ability of MNC2-MMAE to eradicate these same tumors in animals, the attachment of several toxins to the MNC2 antibody may sterically hinder the binding of the secondary antibody.

[0263] One of the key goals that researchers are trying to achieve is to develop an anticancer therapy that can recognize and kill early-stage cancer cells that express low levels of a target antigen. Statistically, cancer patients have a better chance of survival if their cancer is treated at a very early stage. To date, few targeted cancer therapies have been able to kill early-stage, low-antigen-expressing cancer cells. Newer targeted therapies typically can only kill cancer cells when the expression of the target antigen is above a certain threshold. For example, the breast cancer drug Herceptin can only be used if the patient's expression of the target HER2 is relatively high. Even so, about 20% of patients treated with Herceptin develop resistance to Herceptin, which is often accompanied by resistance to other anticancer treatments. It is now recognized that tumor recurrence is often caused by low-antigen-expressing cells that the treatment cannot kill. If the target antigen is a growth factor receptor, then advanced tumors will express higher levels of the growth factor receptor, but these higher levels will also drive the tumor to grow faster than cells that express lower levels of the growth factor receptor.

[0264] Therefore, although it is rarely done, it is critical to test anticancer therapies against early-stage cancer cells that express low levels of the antigen and late-stage cancer cells that express high levels of the targeted antigen. Figures 38A-45DIn the present invention, the ability of MNC2-ADC and MN20A10-ADC to kill multiple cancer subtypes was tested, where the tumors were low-medium antigen expressing cancer cells or high antigen expressing cancer cells. Surprisingly, MNC2-ADC and MN20A10-ADC showed a significant ability to kill both low antigen expressing cancer cells and high antigen expressing cells. Low expressing cancer cells were killed by treatment with MNC2-ADC or MN20A10-ADC, which are considered to be pharmaceutically acceptable ADCs in the high nanomolar range. Cancer cells expressing high levels of MUC1* were killed by IC50s in the single-digit nanomolar range. Submicromolar levels of antibodies are considered pharmaceutically acceptable. Again, since MNC2 and MN20A10 are highly cancer specific, this dose range will be very tolerable. In vitro measurements of ADC killing generally underestimate the in vivo killing potential of ADCs. In vivo, killing due to "bystander" effects is often observed. That is, when the killing of cells within the tumor triggers the host to release cytokines, macrophages, etc., these cytokines, macrophages, etc. will greatly increase the killing. These effects do not occur in vitro.

[0265] For example, MNC2-MMAE, MNC2-MMAF, MN20A10-MMAE, and MN20A10-MMAF were tested 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* in vitro. 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* ( Figure 38A-Figure 38F ). In these experiments, the DARs for MNC2-MMAE and MNC2-MMAF were comparable, at 4.1 and 3.7, respectively. There was a slight difference between the DARs for MN20A10-MMAE and MN20A10-MMAF, at 5.8 and 3.8, respectively, which was reflected in their killing potency. As can be seen in the figure, both wild-type, low antigen expressing cells were killed when treated with submicromolar doses of the ADC that are considered druggable. Surprisingly, although MUC1* is a growth factor receptor, the more MUC1* expressed, the greater the ADC-mediated killing. Both breast and pancreatic cancer cells that were engineered to express more MUC1* were readily killed by MNC2-MMAE, MNC2-MMAF, MN20A10-MMAE, and MN20A10-MMAF at extremely low nanomolar concentrations. The killing of target cells by MNC2-deruteccan or MN20A10-deruteccan was compared with that by MNC2-MMAE, MNC2-MMAF, MN20A10-MMAE, and MN20A10-MMAF ( Fig.40A – Fig.40C In this in vitro assay, all five anti-MUC1* ADCs appeared to have comparable killing potency, although killing of bystander cells could not be determined in this assay. The anti-MUC1*-ADCs were also shown to be MUC1*-positive prostate cancer cells ( Fig.41A –41B) and non-small cell lung cancer cells ( Fig.42A –42B).

[0266] In one set of flow cytometry experiments, the specific killing effect of MNC2-MMAE or MN20A10-MMAE was determined by measuring the viability of target cancer cells after the addition of MUC1*-ADC. The target cells here were T47D wild-type breast cancer cells or T47D breast cancer cells engineered to express more MUC1*, referred to as T47D-MUC1*. As can be seen in Figure 43, both MN20A10-MMAE and MNC2-MMAE were effective in killing wild-type breast cancer cells and cells engineered to express more MUC1*. However, for both MUC1*-ADCs, the killing efficacy was higher when the cells overexpressed the target antigen. Cancer cells expressing low levels of MUC1* are consistent with early-stage cancer, while cells expressing high levels of MUC1* are consistent with advanced cancer.

[0267] To demonstrate that any cell expressing MUC1* will be killed by the MUC1*-ADC described herein, we engineered a MUC1-negative colon cancer cell line, HCT-116, to express MUC1*, such as HCT-MUC1*. As can be seen in the cell viability assay of FIG. 44, only cells expressing MUC1* were killed. The viability of cancer cells expressing MUC1* was measured after adding unconjugated MN20A10 or MN20A10-MMAE. As can be seen in FIG. 45, MN20A10-MMAE effectively kills non-small cell lung cancer cells, DU145 hormone-resistant prostate cancer cells, and pancreatic cancer cells.

[0268] Figures 46-48 show magnified photographs of various types of cancer cells after treatment with MNC2-ADC or MN20A10-ADC. The killing effect can be easily seen as a significant reduction in cell number, a change in cell morphology from a characteristic flat, spread morphology to a rounded morphology and cell shedding. In contrast, the control wells showed a confluent monolayer of dense cells with a normal flat spread morphology and no dead floating cells. Figure 46A-46C and Figure 46G-Figure 46F Shown are T47D-MUC1* breast cancer cells treated with MNC2-MMAE. Figure 46D-Figure 46F and Figure 46G-Figure 46J Shown are T47D-MUC1* breast cancer cells treated with MNC2-derutec. Figure 47A-47BShown are DU145 hormone-resistant prostate cancer cells treated with MNC2-MMAE. Figure 47C-Figure 47D Shown are DU145 hormone-resistant prostate cancer cells treated with MNC2-derutecan. Figure 48A-48B and Figure 48G-Figure 48H Shown are T47D-MUC1* breast cancer cells treated with MN20A10-MMAE. Figure 48C-Figure 48D and Figure 48I-48J Shown are T47D-MUC1* breast cancer cells treated with MN20A10-deruteccan. Figure 48K-48L Shown are DU145 hormone-resistant prostate cancer cells treated with MN20A10-MMAE. Figure 48M-48N DU145 hormone-resistant prostate cancer cells treated with MN20A10-Drutecan are shown. It can be intuitively seen that the killing effect of the anti-MUC1*-ADC shown in Figures 46-48 is consistent with the killing measured by flow cytometry shown in Figures 38-45.

[0269] The ability of MNC2-ADC and MN20A10-ADC to kill both low and high MUC1* expressing cells was also determined by using the xCELLigence instrument to monitor killing in real time (Figures 53-56). In the xCelligence system, adherent target cancer cells are coated onto an electrode array 96-well plate. Adherent cells insulate the electrodes and increase impedance. The number of adherent cancer cells is proportional to the impedance. Antibodies and antibody-drug conjugates are much smaller and do not contribute significantly to impedance. Therefore, an increase in impedance reflects the growth of cancer cells and a decrease in impedance reflects the killing of cancer cells.

[0270] The breast cancer cell line T47D-wt expresses low to low-medium 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 further lower levels of MUC1*. Both MNC2 and MN20A10 conjugated with MMAE, MMAF, derutocan or exatecan can effectively kill 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* are consistent with early cancer cells and 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, in which MNC2-exetatecan (DAR 8.2) was compared with MNC2-drutatecan (DAR 4.0) and MNC2-MMAE (DAR 4.1), MNC2-exetatecan showed more effective killing of cancer cells expressing low MUC1*, which may be due to the higher number of toxins attached thereto.

[0271] In the next set of experiments, the ability of the MNC2-ADC and MN20A10-ADC to kill cancer cells that expressed high levels of MUC1* was tested. The expectation was that the higher the levels of MUC1*, the more difficult it would be to kill the cells, since MUC1* is a growth factor receptor that drives the growth of these cells. Unexpectedly, the experiments showed that the more MUC1* was expressed, the easier it was to kill the cells. The xCELLigence real-time killing assay was also used to compare the killing of cancer cells that expressed higher levels of MUC1* by MNC2 and MN20A10 conjugated to MMAE, MMAF, derutocan, or exatecan. The breast cancer cell lines T47D-MUC1* and HPAF II-MUC1* have been engineered to express high levels of MUC1*, consistent with advanced cancer. As can be seen in Figures 50, 53, and 55, cancer cells that express high levels of MUC1* are completely killed at very low nanomolar doses (6nM–19nM). S

[0272] In addition to the in vitro assays, in vivo experiments were performed in which the anti-MUC1* antibodies MNC2 and MN20A10 conjugated to MMAE, MMAF, derutocan, or exatecan were administered to test mice implanted with breast, lung, or pancreatic tumors expressing high or low MUC1*. These experiments are described in more detail in Experiment 18. In one example, NOD / SCID / GAMMA mice were implanted with T47D breast cancer cells that were either wild type or engineered to express more MUC1*. The animals were given three injections of MNC2-MMAE (DAR 3.85) at an initial dose of 5 mg / kg, which was then increased to 10 mg / kg. As can be seen in Figure 57, the tumors carried by the mice expressing high levels of MUC1* (consistent with advanced cancer) were effectively eliminated at day 26. The tumors implanted in the mice with cells expressing low MUC1* (consistent with early cancer) were not completely eliminated, but the tumor size was smaller compared to the control group. In contrast (Figure 58), animals given the same dose of MN20A10-MMAE (DAR 2.96) did not have complete tumor elimination, likely due to the lower number of attached toxins. Animals implanted with non-small cell lung cancer tumors were given the same dose of MNC2-MMAE (Figure 59) or MN20A10-MMAE (Figure 60). MNC2-MMAE (DAR 4.1) was very effective in eliminating tumors in test mice implanted with pancreatic tumors with high or low MUC1* expression (Figure 61). Figure 62 shows an update of the experiment shown in Figure 61. After 66 days, animals treated with MNC2-MMAE were tumor-free since day 18, with no tumor recurrence. MNC2-Drutecan was administered to mice implanted with human breast cancer cells expressing low or high levels of MUC1* (Figures 63 and 64). Tumors expressing high MUC1* were essentially eliminated on day 26. Mice implanted with significantly fewer cells expressing low MUC1* compared to the control group, but higher doses or repeated injections may be required.

[0273] In animal models, we show that as tumors progress to advanced stages, their expression of MUC1* increases. Figure 65A-Figure 65D Images of tumor cells are shown. Staining of day 0 cells shows that the cells express more full-length MUC1 than MUC1*, and the staining intensity is lighter, indicating a low number of MUC1* receptors, which is indicative of an early stage cancer. Sixty-two (62) days after tumor implantation, which is equivalent to approximately 7 years in human time, it can be easily seen that MUC1* expression (in terms of expression level and expression intensity) has significantly shifted from low expression to high expression in advanced tumors. In contrast, staining of serial sections of the tumor showed that full-length MUC1 was expressed as expected, because it is cleaved into MUC1* after surface expression. However, the staining intensity did not increase, indicating that the vast majority of the MUC1 expressed in advanced tumors has been cleaved into the growth factor receptor form MUC1*.

[0274] Antibody

[0275] Those skilled in the art will understand that in this context, the antibody (Ab) that binds to the target polypeptide binds to "binding". For example, an antibody or a conjugate described herein comprising such an Ab may bind to other polypeptides or proteins, usually with lower affinity, as determined by, for example, an immunoassay or other assay known in the art. In a specific embodiment, the Ab that specifically binds to the target polypeptide or a conjugate described herein comprising such an Ab binds to the target polypeptide with an affinity that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater of the affinity of the Ab or conjugate when conjugated to another polypeptide. In another specific embodiment, the Ab or a conjugate described herein comprising such an Ab does not specifically bind to a polypeptide other than the target polypeptide. In a specific embodiment, the Ab or a conjugate described herein comprising the Ab specifically binds to the target polypeptide with an affinity (Kd) less than or equal to 20 mM. In certain embodiments, such binding has an affinity (Kd) of less than or equal to about 20 mM, about 10 mM, about 1 mM, about 100 μM, about 10 μM, about 1 μM, about 100 nM, about 10 nM, or about 1 nM. Unless otherwise indicated, "bind", "binds to", "specifically binds to", or "specifically binds to" are used interchangeably in this context.

[0276] In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer is breast cancer, colon cancer, prostate cancer, pancreatic cancer, or lung cancer.

[0277] In some embodiments, the antibody binds to cancer cells that express MUCl* on their surface.

[0278] 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.

[0279] 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.

[0280] In certain embodiments, the conjugate comprises an antibody Ab. In certain embodiments, Ab is a monoclonal antibody. In certain embodiments, Ab is a human antibody. In certain embodiments, Ab is a humanized antibody. In certain embodiments, Ab is a chimeric antibody. In certain embodiments, Ab is a full-length antibody comprising two heavy chains and two light chains. In specific embodiments, Ab is an IgG antibody, such as an IgG1, IgG2, IgG3 or IgG4 antibody. In certain embodiments, Ab is a single-chain antibody. In other embodiments, Ab is an antigen-binding fragment of an antibody, such as a Fab fragment.

[0281] In a specific embodiment, Ab is an IgG1 antibody. In a specific embodiment, Ab is an IgG2b antibody.

[0282] In certain embodiments, the antibodies specifically bind to cell surface proteins. In certain embodiments, the antibodies specifically bind to cell surface receptors. In certain embodiments, the antibodies specifically bind to cell surface receptor ligands.

[0283] In some embodiments, the antibody or a functional fragment or functional variant thereof specifically binds to MUCl *. In some embodiments, the antibody comprises an anti-MUCl * heavy chain and an anti-MUCl * light chain.

[0284] 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 a 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 a variable domain of a kappa or lambda light chain.

[0285] 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 κ or λ 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 κ or λ 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 κ or λ 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 κ or λ light chain.

[0286] 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.

[0287] 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.

[0288] In some embodiments, the antibody or its functional fragment or functional variant that specifically binds to MUC1* comprises a single chain variable fragment (scFv) or an antigen binding fragment (Fab). In some embodiments, the antibody or its functional fragment or functional variant that specifically binds to MUC1* comprises a single chain variable fragment. In some embodiments, the antibody or its functional fragment or functional variant that specifically binds to MUC1* comprises an antigen binding fragment (Fab).

[0289] In some embodiments, the anti-MUC1* heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the anti-MUC1* heavy chain variable domain comprise an amino acid sequence according to: HC-CDR1: SEQ ID NO: 1 or 4; HC-CDR2: SEQ ID NO: 2 or 5; HC-CDR3: SEQ ID NO: 3 or 6; and wherein the CDRs comprise 0-2 amino acid modifications (e.g., 0 or 1 amino acid modification) in at least one of HC-CDR1, HC-CDR2, or HC-CDR3.

[0290] In some embodiments, the anti-MUC1* light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the anti-MUC1* light chain variable domain comprise an amino acid sequence according to: LC-CDR1: SEQ ID NO: 13 or 16; LC-CDR2: SEQ ID NO: 14 or 17; LC-CDR3: SEQ ID NO: 15 or 18; and wherein the CDRs comprise 0-2 amino acid modifications (e.g., 0 or 1 amino acid modifications) in at least one of LC-CDR1, LC-CDR2, or LC-CDR3.

[0291] In some embodiments, the anti-MUCl* heavy chain comprises an amino acid sequence 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% identical to SEQ ID NO:38 or 44. In some embodiments, the anti-MUCl* light chain comprises an amino acid sequence 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% identical to SEQ ID NO:41 or 47.

[0292] Antibody drug conjugates (ADCs) work when the variable region of the antibody portion recognizes a molecule expressed externally on cancer cells. After the antibody binds to the target antigen, the entire ADC is internalized. Antibody internalization is a requirement for the cell-killing function of the ADC. Often, cellular internalization of the ADC biochemically alters the toxin, in some cases making it more effective, while in other cases this alteration traps the toxin inside the cell to reduce off-target toxicity. Not all receptors are internalized upon ligand binding, or particularly upon antibody binding, which dimerizes the receptor, making internalization more difficult or even impossible. Fig.25A -25D shows a photograph taken on a confocal microscope documenting the internalization of an anti-MUC1* antibody (eg, MNC2) after binding to the extracellular domain of the MUC1* receptor.

[0293] Payload

[0294] Antibody drug conjugates (ADCs) combine the target specificity of antibodies (e.g., monoclonal antibodies) with the potency of small molecule drugs (called payloads or cytotoxic groups), connecting them to a single ADC molecule that retains the properties of both. Compared with traditional chemotherapy drugs, ADCs have improved selectivity and potency, leading to greater safety and efficacy, resulting in a wider therapeutic window. The terms "payload" and "cytotoxic group" are used interchangeably in this article.

[0295] 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 derutecan or a derivative thereof.

[0296] 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).

[0297] Some of the newer toxic payloads used in ADC formats, such as delutec, belong to the exatecan family of topoisomerase I inhibitors. One such new payload-coupler configuration used in ADC formats is delutec, such as Fig.28 Here, the maleimidocaproyl (MC) moiety facilitates coupling to a cysteine ​​on the antibody. The maleimidocaproyl group is linked to the toxic payload Dxd via a glycine-phenylalanine linker GGFG, a coupling agent HN-CH2- to Dxd. Dxd is a topoisomerase I inhibitor, and this is the mechanism by which it inhibits cell division. Fig.29 In another example shown, the exatecan payload is attached to the antibody via a p-aminobenzyl (PAB) moiety, which is linked to a valine-citrulline (VC) moiety, which is in turn linked to a maleimidocaproyl (MC) moiety that facilitates coupling to cysteines on the antibody.

[0298] Exatecans have high levels of bystander effects, meaning they can kill neighboring cells. Clinical trials of the new ADC desartans (Enhertu) targeting HER2+ breast cancer were plagued by severe and life-threatening side effects such as pneumonitis. Up to 16% of desartans patients reportedly suffered from treatment-induced pneumonitis. However, desartans did receive FDA approval because it reportedly extended survival by nearly two years in patients with metastatic breast cancer. It is not entirely clear whether these side effects were due to the payload or the target HER2, which is also expressed on 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 suggested that the deaths were caused by the use of an antibody with extremely high affinity for HER2. Recently, another patient died after being treated with a different HER2-targeted ADC. These results highlight the importance of selecting antibodies with excellent cancer selectivity to avoid these life-threatening toxicities. MNC2 and MN20A10 unexpectedly have extremely high cancer specificity and did not cause toxicity in animal studies in which each antibody was incorporated into several ADC formats.

[0299] Connectors

[0300] In certain embodiments, the joint-L- comprises one or more of a carbon atom, a nitrogen atom, a sulfur atom, an oxygen atom, and a combination thereof. In certain embodiments, the joint-L- comprises one or more amino acids. In certain embodiments, the joint-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 a combination thereof. In certain embodiments, the joint-L- comprises a linear structure. In certain embodiments, the joint-L- comprises a dipeptide, a tripeptide, or a tetrapeptide connecting portion.

[0301] The linker may comprise at least one glycine. The linker may comprise at least one glycine and phenylalanine. The linker may comprise the structure The linker may comprise valine. The linker may comprise citrulline. The linker may comprise valine and citrulline. The linker may have the structure The dipeptide linking portion.

[0302] In certain embodiments, Z is a conjugate moiety capable of forming a covalent bond with an amino acid of a polypeptide. Z may be bound to the N-terminus of a linker. Z may include maleimide. In certain embodiments, the amino acid is cysteine. In certain embodiments, Z is In certain embodiments, Z is In certain embodiments, Z is

[0303] The linker may comprise a coupling moiety R. In certain embodiments, R is a coupling moiety capable of binding a payload to the C-terminus of the linker. R may comprise a moiety having the structure where * indicates the attachment point of the payload. R can contain structural groups where * indicates the attachment point of the payload. R can contain a Wherein * represents the attachment point of the payload (e.g., cytotoxic group). R may include a group having the structure where * indicates the attachment point of the payload.

[0304] Fig.26 The chemical structure of the payload monomethyl auristatin E (MMAE) is shown, along with convenient linker molecules and reactive molecules to facilitate chemical coupling of the payload to the antibody. In this case, MMAE is linked to the antibody via a p-aminobenzyl (PAB) moiety, which is linked to a valine-citrulline (VC) moiety, which in turn is linked to a maleimidocaproyl (MC) moiety that facilitates coupling to cysteines on the antibody. Once 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.

[0305] Monomethyl auristatin F, known as MMAF, is another example of a toxic payload that inhibits cell division by blocking the polymerization of tubulin. Fig. 27 In the example shown, the payload MMAF is linked to the antibody via a p-aminobenzyl (PAB) moiety, which is linked to a valine-citrulline (VC) moiety, which in turn is linked to a maleimidocaproyl (MC) moiety that facilitates coupling to cysteines on the antibody. Once the ADC is internalized, cathepsin B enzymatically cleaves the payload from the antibody. Unlike MMAE, MMAF contains a carboxylic acid, making it difficult for the payload to be excreted from the cell after cleavage from the antibody.

[0306] Some of the newer toxic payloads used in ADC formats, such as delutec, belong to the exatecan family of topoisomerase I inhibitors. One such new payload-coupler configuration used in ADC formats is delutec, such as Fig.28 A– Fig.28E. Here, the maleimidocaproyl (MC) moiety facilitates coupling to a cysteine ​​on the antibody. The maleimidocaproyl group is linked to the toxic payload Dxd via a glycine-phenylalanine linker GGFG, a coupling agent HN-CH2- to Dxd. Dxd is a topoisomerase I inhibitor, and this is the mechanism by which it inhibits cell division. Fig.29 A- Fig.29 In another example shown in E, we attached the exatecan payload to our antibody via a p-aminobenzyl (PAB) moiety, which was linked to a valine-citrulline (VC) moiety, which in turn was linked to a maleimidocaproyl (MC) moiety that facilitated coupling to cysteines on the antibody.

[0307] Pharmaceutical composition

[0308] In another aspect, provided herein are pharmaceutical compositions comprising a conjugate disclosed herein (e.g., ADC) and a multispecific antibody. In some embodiments, the pharmaceutical composition comprises a conjugate of formula (I) and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a multispecific antibody disclosed herein and a pharmaceutically acceptable carrier.

[0309] The pharmaceutical compositions herein are formulated with one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active agents into preparations which can be used pharmaceutically. Proper formulation depends upon the route of administration chosen.

[0310] In certain embodiments, the pharmaceutical compositions disclosed herein further comprise a pharmaceutically acceptable diluent, excipient or carrier. In some embodiments, the pharmaceutical compositions include other medicinal or pharmaceutical agents, carriers, adjuvants, such as preservatives, stabilizers, wetting agents or emulsifiers, dissolution promoters, salts for regulating osmotic pressure and / or buffers.

[0311] 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.

[0312] Preparations suitable for intramuscular, subcutaneous, peri-tumor 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.), their suitable mixtures, vegetable oils (e.g. olive oil) and injectable organic esters, such as ethyl oleate. The maintenance of fluidity is achieved, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion and by using a surfactant. Preparations suitable for subcutaneous injection also include optional additives, such as preservatives, wetting agents, emulsifiers and dispersants.

[0313] For intravenous injection, the active agent is optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.

[0314] Parenteral injections may optionally include push injections or continuous infusions. Injectable formulations may optionally be in unit dose form, such as in ampoules or multidose containers, and preservatives may be added. In some embodiments, the pharmaceutical compositions described herein are in a form suitable for parenteral injection, as a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and contain a preparatant, such as a suspending agent, a stabilizer and / or a dispersant. Pharmaceutical formulations for parenteral administration include aqueous solutions of active agents in water-soluble form. In addition, the suspension may optionally be prepared as a suitable oily injection suspension.

[0315] In some embodiments, the pharmaceutical composition described herein is a unit dosage form suitable for single administration of precise dosage. In the unit dosage form, the preparation is divided into unit doses containing an appropriate amount of the active agent disclosed herein. In some embodiments, the unit dose is a packaged form comprising a discrete amount of preparation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. In some embodiments, the aqueous suspension composition is packaged in a single dose non-reclosable container. Alternatively, a multi-dose reclosable container is used, in which case a preservative is generally included in the composition. For example only, the preparation for parenteral injection is presented in a unit dosage form (including but not limited to ampoules) or in a multi-dose container (with an added preservative).

[0316] Example

[0317] Example 1. Anti-MUC1* internalization assay

[0318] Confirmed that MNC2 internalization ( Figure 25A-25B ).

[0319] 8-well chamber slide (Nunc TM Lab-Tek TM IIChamber Slide TM System Thermo Part #154534PK or Ibidi TM 8-well μ-slides (ibiTreat: #1.5 polymer coverslips, tissue culture treated, sterilized) were coated with collagen (Sigma #C3867) where 300 μL collagen was added to each well and incubated overnight at 4°C, washed once with PBS and then once with water, and then air-dried in a biosafety hood. The wells were dried under the hood.

[0320] T47D breast cancer cells were seeded at 30,000-50,000 cells per well in 10% RPMI and cultured for 48 hours or until ~70% confluent. The medium was removed and the cells were serum starved for 24 hours in 2% RPMI. The cells were washed with ice-cold PBS.

[0321] Antibodies were diluted in cold PBS plus 2% FBS to a final concentration of 300ug / mL. 200ul of antibody solution was added to each well and incubated at 4°C in the dark for 2 hours on a rocking platform shaker at speed 6. Cells were then washed three times with cold PBS.

[0322] 10ug / mL of anti-mouse Alexa 488 antibody or anti-human Alexa 555 antibody was diluted 1:200 in PBS containing 2% FBS and incubated with shaking for 1 hour in the dark at 4°C. The cells were washed three times with cold PBS.

[0323] For internalization, warm 10% RPMI + 75 nM lysotrackerdeep red (1 / 13,333) was added and incubated for 2 hours in a CO2 incubator at 37°C. The cells were washed three times with cold PBS.

[0324] Fix cells with fresh 4% formaldehyde in PBS for 15 minutes at room temperature and then wash three times with cold PBS. Add 1 ug / mL of Hoescht dye 33342 in PBS and incubate at room temperature for 10 minutes, then remove the medium without washing steps. TM Lab-Tek TM , mount the cover slip (No. 1.5) on the chamber slide with mounting medium (Prolong Diamond antifade), avoiding air bubbles. Then store the slide on a horizontal surface at room temperature for 24 hours. Long-term storage at 4°C in the dark. For Ibidi slides, add Ibidi mounting medium.

[0325] Photographs were taken on a confocal microscope.

[0326] Example 2. General description of small-scale (3 mg) preparation of MNC2-ADC or MN20A10-ADC (antibody-drug-conjugate)

[0327] A solution of unconjugated MNC2 (IgG1 antibody) or MN20A10 (IgG2b antibody) was reduced with excess dithiothreitol (DTT) in a pH 8.0 borate buffer. After reduction at 37°C for 2 hours, the amount of free thiols present in the aliquots was tested using the Ellman thiol test. The number of free thiols will vary depending on the antibody isotype. If analysis of free thiols using Ellman's reagent indicates an insufficient number of free thiols, more DTT is added and incubated at 37°C for an additional period of time. The process of free thiol determination and addition of more reducing agent is repeated until a sufficient number of free thiols is obtained. MN20A10 requires more reducing agent than MNC2 and the reduction time is longer than MNC2. The reduced antibody is then placed in a pH 6.0 2-(N-morpholino)ethanesulfonic acid (MES) buffer and the free cysteine ​​is alkylated with 11 molar equivalents of a maleimide-linked toxic agent for about 1 hour. The crude ADC is then purified by desalting columns to remove low molecular weight contaminants such as excess toxic agents, DMSO and other small molecular weight contaminants. The drug-antibody ratio (DAR) is calculated based on the ratio of toxin UV absorbance @ 248 nm (or 370 nm in the case of derutocan) to antibody absorbance @ 280 nm. According to the literature method (Effects of drug loading on the 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), the conjugates were analyzed by hydrophobic interaction chromatography-high performance liquid chromatography to determine the drug-antibody ratio (DAR), i.e., the [drug:mAb] molar ratio.

[0328] Small scale preparations of MNC2-MMAE, MNC2-MMAF, MNC2-drutecan, MNC2-exetatecan, MN20A10-MMAE, MN20A10-MMAF, MN20A10-drutecan, and MN20A10-exetatecan were generated following this protocol.

[0329] Example 3: Small scale (3 mg) preparation of MNC2-drunotecan Detailed example of generation

[0330] IgG1 MNC2 antibody (3.00 mg, 1.578 ml, 1.9 mg / ml, 20 nM) was thawed from -80°C and placed in a 37°C water bath for 1 minute. The protein concentration of mouse IgG1 antibody 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 cutoff (Cat. #UFC505024) (14,000xg, 8 minutes at 4°C) to obtain a volume equal to or less than 100 μL. The buffer was then exchanged to pH 8.0 borate buffer (25mM sodium tetraborate, 25mM NaCl, 1mM EDTA) by concentrating to <100 μL and resuspending in approximately 300 μL of fresh buffer. This process was repeated a total of 3 times. The concentrated antibody (~100uL) was transferred to a 0.5mL screw cap centrifuge tube and brought to 300μL with pH 8.0 borate buffer (25mM sodium tetraborate, 25mM sodium chloride, 1mM EDTA) to prepare a 10mg / mL antibody solution.

[0331] Dithiothreitol (DTT) (6.48mM) solution was prepared by weighing DTT (5mg, 32.4mmol) and dissolving it in 5mL of pH 8.0 borate buffer (25mM sodium tetraborate, 25mM sodium chloride, 1mM EDTA). DTT solution (6.48mM, 30.9uL, 200nmol, 10 equivalents) 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 regularly every 15 minutes. After 2 hours, an aliquot (6uL) of the reaction mixture was taken out for Ellman thiol analysis, which showed that each antibody had 7.68 free thiols, while IgG1 may have a total of 8 thiols.

[0332] The antibody reduction mixture was cooled to 4°C in an ice bath and the solution was transferred to a centrifugal filter (AmiconUltra 0.5mL 50,000mw cutoff) and the buffer was switched 3 times to pH 6.0 MES buffer (50mM). [14,000xg, 8 minutes at 4C). The concentrated reduced antibody was diluted to 640μL with pH 6.0 MES buffer (50mM).

[0333] A stock solution of derutecan (10 mg / mL, 9.7 mM) in DMSO was prepared by weighing 1.80 mg, 1.74 μMole and dissolving it in DMSO (180 uL). A solution of derutecan (2 mM, 110.2 uL, 220 nmole) in 50% DMSO / pH 6.0 MES buffer (50 mM) was prepared by diluting the derutecan 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 prepare a 2 mM derutecan solution in 50% DMSO / 50% pH 6.0 MES buffer (50 mM). The derutecan solution (110.1 uL, 50% DMSO / 50% pH 6.0 MES buffer, 2 mM, 73.4 nmole, 11 equivalents) was added to a tube containing 640 μL of reduced antibody. The reaction was slowly mixed on a vertical rotator at room temperature for 1 hour and monitored by hydrophobic interaction chromatography (HIC) HPLC, which indicates whether there is any unreacted antibody remaining. The retention time of trace amounts of unreacted antibody is compared with the reaction product to determine whether unreacted antibody remains, and if so, the reaction is allowed to continue. For small scale, the time is typically 1-2 hours. Large-scale preparations may require allowing the reaction to proceed within 8-12 hours.

[0334] After about 1 hour, the reaction was purified on a desalting column (Cytiva PD miditrap g-25 medium, catalog #28918008). The top of the column was opened and the packing buffer was discarded. The bottom of the column was opened and the column was conditioned by gravity flow through with Gibco PBS pH 7.4 (reference catalog #10010-023) (3x 5mL). The antibody solution (750uL) was loaded onto the column and allowed to fully enter the resin bed, and then PBS (250uL) was added. The purified ADC was then eluted from the column with pH 7.4PBS (1.25mL). Nanodrop protein concentration determination showed that the antibody in 1.25mL of PBS was 1.76mg / mL, or 2.2mg (yield of 73%) conjugated antibody was recovered from the initial 3.0mg of unconjugated antibody. The purified ADC was analyzed by UV-VIS spectroscopy at 370nm and 280nm, giving a drug-antibody ratio (DAR) of 6.7.

[0335] Example 4: MN20A10-Drutecan

[0336] MN20A10-drutecan was prepared in a similar manner but using 20 equivalents of DTT which gave 4.77 free thiols per antibody. After conjugation with drutecan, the purified ADC had a DAR of 4.60 as measured by UV-VIS spectroscopy at 370 nm and 280 nm.

[0337] Example 5: MNC2-MC-VC-PAB-MMAE

[0338] MNC2-MC-VC-PAB-MMAE was prepared in a similar manner, but using 5 equivalents of DTT, which gave 6.12 free thiols per antibody. After coupling with MC-VC-PAB-MMAE, the purified ADC had a DAR of 4.39 as measured by HIC-HPLC and a DAR of 5.12 as measured by UV-VIS spectroscopy at 248 nm and 280 nm.

[0339] Example 6: MNC2-MC-VC-PAB-MMAF

[0340] MNC2-MC-VC-PAB-MMAF was prepared in a similar manner, but using 5 equivalents of DTT, which gave 4.53 free thiols per antibody. After coupling with MC-VC-PAB-MMAF, the purified ADC had a DAR of 3.65 as measured by HIC-HPLC.

[0341] Example 7: MNC2-MC-VC-PAB-exatecan

[0342] MNC2-MC-VC-PAB-exatecan was prepared in a similar manner, but using 10 equivalents of DTT, which gave 6.20 free thiols per antibody. After conjugation with MC-VC-PAB-exatecan, the purified ADC had a DAR of 8.40 measured by UV-VIS spectroscopy at 370 and 280 nm.

[0343] Example 8: MN20A10-MC-VC-PAB-MMAE

[0344] MN20A10-MC-VC-PAB-MMAE was prepared in a similar manner, but using 7.5 equivalents of DTT, which gave 4.08 free thiols per antibody. After coupling with MC-VC-PAB-MMAE, the purified ADC had a DAR of 3.81 as measured by HIC-HPLC and a DAR of 4.11 as measured by UV-VIS spectroscopy at 248 and 280 nm.

[0345] Example 9: MN20A10-MC-VC-PAB-MMAF

[0346] MN20A10-MC-VC-PAB-MMAF was prepared in a similar manner, but using 15 equivalents of DTT, which gave 3.87 free thiols per antibody. After coupling with MC-VC-PAB-MMAF, the purified ADC had a DAR of 3.79 as measured by HIC-HPLC.

[0347] Example 10 Large-scale (30 mg) preparation of MNC2-ADC or MN20A10-ADC antibody-drug-conjugate.

[0348] In a similar manner to the preparation of small-scale ADC, the unconjugated antibody solution was reduced with excess dithiothreitol (DTT) in pH 8.0 borate buffer. After reduction for 2 hours, the amount of free thiol present in the aliquot was tested using the Ellman thiol test method. The reduced antibody was then placed in pH 6.0 MES buffer, and the free cysteine ​​was alkylated overnight with 11 molar equivalents of maleimide-linked toxic agents. The crude ADC was then purified by a desalting column.

[0349] Aliquots of IgG1 MNC2 antibody (15.7 mL of 1.92 mg / mL, 30.1 mg, 200 nmole) were thawed from -80°C and placed in a 37°C water bath for 1 minute. The volumes were combined and the concentration was determined by nanodrop using the mouse IgG1 antibody setting (background subtraction turned off). 30 mg of antibody in 16.17 mL was 1.9 mg / mL, 200 nmol. The solution was concentrated using 3 centrifugal filters (Amicon Ultra 4 mL 30,000 mw cutoff (reference number # UFC803024)) and the buffer was changed to pH 8.0 borate buffer (25 mM sodium tetraborate, 25 mM NaCl, 1 mM EDTA) and spun 3 times at 4000 g for 15-25 minutes in an Eppendorf 5804R centrifuge with a swing-out 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.

[0350] DTT solution (6.48 mM) was prepared by weighing 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). DTT solution (6.48 mM, 617 uL, 4000 nmole, 20 equivalents) was added to the antibody solution and the tube was heated at 37°C in a shaking incubator for 2 hours. After 2 hours, an aliquot (6 uL) of the reaction mixture was removed for Ellman thiol analysis. Analysis of these results showed 7.12 moles of free thiol per mole of antibody.

[0351] In a similar manner, MN20A10 was reduced with a total of 20 equivalents of DTT during 3 hours and multiple free thiol assays were performed.

[0352] The reduced antibody tube was then cooled to 4C in an ice bath, the solution was transferred equally to 3 centrifugal filters (AmiconUltra 4mL 30,000 to mw cutoff (reference number # UFC803024)), and the buffer was exchanged to pH 6.0 MES (50mM) buffer, spun three times at 4000g for 15-25 minutes in an Eppendorf 5804R centrifuge. The concentrate was transferred to a 15mL falcon tube, and the concentrator was rinsed twice with buffer, and the final volume was brought to 6400μL with pH6.0 MES (50mM) buffer.

[0353] A stock solution of derutecan (10 mg / mL, 9.67 mM) in DMSO was prepared by weighing derutecan (3.4 mg, 3.29 μMol) and dissolving it in DMSO (340 uL). 1100 μL of a 2 mM derutecan solution in 50% DMSO / pH 6.0 MES (50 mM) was prepared by diluting the DMSO stock derutecan solution (227.5 μL of 9.67 mM, 2.22 μMole) with DMSO (322.5 uL) and then further diluting with pH 6.0 MES (50 mM, 550 uL) to make 1100 μL of 2.068 mg / mL (2 mM) derutecan in 50% DMSO / 50% pH 6.0 MES (50 mM). Derutecan solution (2 mM, 1000 uL, 2200 nmole, 11 equiv) was added to the tube containing 6400 μL of reduced antibody. The reaction was mixed on a vertical rotator at room temperature for 18 hours and monitored by HIC HPLC after 1 hour and the next day.

[0354] After 18 hours, the reaction was purified on a desalting column (Cytiva Sephadex G-25 medium catalog #17003301). Sephadex g-25 intermediate gel (10.13 g) was expanded in Gibco PBS pH 7.4 (Gibco catalog #10010-023, 50 mL) in a round bottom flask at room temperature for 3 hours. 25 g of Teledyne Isco Redi-Sep sample loading box (Isco catalog #693873240) was attached to the ring stand and PBS was injected through the bottom to start the box. After the resin expanded for 3 hours, the flask was vacuum degassed 3 times with argon / vacuum. The expanded resin was then poured into the column and the bottom was opened to allow PBS to elute. The round bottom flask was washed with PBS to transfer all the resin to the box. A waste container was placed under the column. The column was conditioned by gravity flow through Gibco PBS pH 7.4 (reference number # 10010-023) 3x 50mL. The filter was placed on top of the box and the column packing tool was pushed down, paying attention to only slightly compressing the gel. The column height was about 74mm. The antibody solution (7500uL) was loaded onto the column and allowed to fully enter the resin bed. PBS (2500uL) was added to the column. A new 50mL falcon tube was placed under the column and the purified ADC was eluted with PBS pH 7.4 (14mL). In order to improve the column efficiency, 1mL fractions were passed through the column until a total of 50mL passed through the column. The collected solution was analyzed with nanodrop to show that the antibody concentration in 17.5mL PBS was 1mg / mL, or 17.5mg antibody, and the yield from unconjugated MNC2 was 58%.

[0355] Example 11: MNC2-MMAE

[0356] MNC2-MMAE was prepared in a similar manner, but using 10 equivalents of DTT, which gave 6.32 free thiols per antibody. After coupling with MC-VC-PAB-MMAE, the purified ADC had a DAR of 4.10 as measured by HIC-HPLC and a DAR of 5.20 as measured by UV-VIS spectroscopy at 248 nm and 280 nm.

[0357] Example 12: MN20A10-MMAE

[0358] MN20A10-MMAE was prepared in a similar manner but using 20 equivalents of DTT, which gave 3.67 free thiols per antibody. After coupling with MC-VC-PAB-MMAE, the purified ADC had a DAR of 2.96 as measured by HIC-HPLC and a DAR of 3.57 as measured by UV-VIS spectroscopy at 248 nm and 280 nm.

[0359] Table 8: ADC DAR

[0360]

[0361] In general, MN20A10 required unexpectedly large amounts of reducing agent (DTT) to obtain the same number of free thiols compared to the MNC2 antibody.

[0362] MN20A10 required 20 equivalents of DTT to obtain 3.67-4.2 free thiols (Examples 11 and 3).

[0363] Scaling up MN20A10 using 20 equivalents of DTT produced less free thiol (3.67) than the small scale (4.2).

[0364] Examples 7 and 8 demonstrate the challenges associated with reducing MN20A10.

[0365] In Example 7, 7.5 equivalents of DTT gave 4.08 free thiols, while in Example 8, even more (15 equivalents) was used to produce similar or lower reduction (3.87 free thiols).

[0366] In contrast, MNC2 required only 5 equivalents of DTT to yield 4.5-6.1 free thiols (Examples 5 and 4).

[0367] 20 equivalents of DTT were used on MNC2-delutecan (3 mg and 30 mg) to force the reaction to the highest possible DAR. (Examples 2 and 9).

[0368] MNC2-MC-VC-PAB-Exactacan (Example 6) was reduced with 10 equivalents of DTT to directly compare the results with Example 2.

[0369] Example 13: Ellman's Thiol Test for Determining the Number of Free Thiols in ADC Reduction

[0370] Prepare 40 mL of 2 mM Ellman's reagent solution by dissolving 31.7 mg of Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid, ThermoScientific Catalog #22582) and 164 mg of sodium acetate in 40 mL of water. Store Ellman's solution at 4C.

[0371] Desalt Reduced Antibody Aliquots: Take two spin columns (Zeba Spin Desalting Columns Reference #89882) and snap off the bottoms and loosen the caps. Place them into spin centrifuge tubes. Spin at 1.5xg for 1 minute in a microcentrifuge (Spectrafuge 24D) to remove the buffer. Discard the eluted buffer. Condition the columns by adding 400 μL of buffer (pH 8.0 borate buffer, composed of 25mM sodium tetraborate, 25mM NaCl, 1mM EDTA) for 5 minutes, then centrifuge at 1.5xg for 1 minute. Discard the eluate between spins. Repeat conditioning and centrifugation.

[0372] Prepare Ellman's Reagent: To prepare 1 mL of Ellman's reagent, pipette 850 μL of water into a tube. Add 100 μL of 1M Tris-HCl pH 8.0, then add 50 μL of Ellman's stock solution prepared above. Add 70 μL to the bottom of two new centrifuge tubes (Protein LoBind Tube, Eppendorf Cat. #022431102), one labeled sample and the other labeled control. After conditioning the columns, move them to clean new centrifuge tubes labeled sample and control.

[0373] Sample and Control Preparation: The final concentration of thiols 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 5x dilutions, which bring the concentration to 13.3 μM. Based on past data, a reading of ~0.24 mAu would correspond to ~4 thiols per antibody when the control is ~0.03. If more equivalents of reducing agent are added, the new concentration should be reflected in the next thiol test.

[0374] Prepare the control solution just before running the test. The control should have the same DTT or TCEP concentration as the reaction without antibody present. Pipette 24 μL of borate buffer (or appropriate buffer) into two tubes labeled Sample Preparation and Control Preparation. Add 6 μL of reduced antibody solution and 6 μL of control solution to each tube respectively. Slowly load 30 μL from each tube into the corresponding tube with the desalting column. Try to distribute the liquid to the middle of the desalting resin without touching the sides. Spin the test tube at 1.5g for 2 minutes and analyze the flow-through in 100 μL cuvette on a spectrophotometer at 412nm.

[0375] The antibody concentration in the thiol test is calculated by using C1 x V1 = MNC2 x V2. Where C1 is the initial concentration (66.7 μM), V1 is the 6 μL taken from the sample, and V2 is the 30 μL it was diluted to. Solving for MNC2 gives the concentration of the antibody in the thiol assay, which is 13.34 μM. The absorbance at 412 nm is used to determine the concentration of free thiols in the sample. First, subtract the absorbance of the control from the sample. The absorbance minus the control is then substituted into Beer's law A = ebc, where A is the absorbance, e is the molar absorptivity of the antibody, b is the path length of the cuvette, and c is the molar concentration. The molar absorptivity 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 thiols in the sample.

[0376] Once the concentration is determined, the dilution factor from 30 μL passed through the column to 70 μL of Ellman's reagent is 3.333. The dilution-adjusted thiol concentration is divided by the antibody concentration to calculate the number of free thiols per antibody.

[0377] Example 14: Determination of Drug-Antibody Ratio (DAR) by UV

[0378] To estimate DAR, the ratio (R) of the UV absorbance of the ADC at 248 nm and 280 nm was measured in a quartz cuvette. R = (absorbance @ 248 nm) / (absorbance @ 280 nm).

[0379] Substitute the R value into the following formula and calculate the DAR: DAR = (21 x R - 9) / (1.615 - 0.1425 x R).

[0380] Example 15: Determination of Drug-Antibody Ratio (DAR) by HIC-HPLC

[0381] Another method that can be used to estimate the DAR of an ADC is to use HIC-HPLC analysis. Briefly, samples of ADC and unconjugated antibody are injected into the HPLC. Peaks observed at different retention times correspond to different drug-antibody ratios. HPLC retention times and peak areas are analyzed in a spreadsheet, where the peaks are initially arbitrarily set to DARs of 1-8 for IgG1 and 1-10 for IgG2. The times between peaks are used in conjunction with the analyst's attempt to fit these peaks to integers corresponding to the number of specific drugs bound to the antibody. This process is complicated by the fact that there are several different ADCs for each DAR. For example, there are 8 possible ACDs (maleimide binding sites) for a DAR of 1. Typically, the retention time differences between different DARs are greater than the retention time differences within a group of ACDs with the same DAR. After the analyst sets the DAR for each peak, the peak areas are added together and the contribution of each DAR peak area is calculated. The average DAR of the sample is then determined based on all peaks.

[0382] Drug-antibody ratio (DAR) and drug loading distribution were determined by hydrophobic interaction chromatography and reversed-phase HPLC, Jun Ouyan, Methods Mol. Biol. 2013; 1045: 275-83.

[0383] Example 16. Measuring the killing of target cells by MNC2-ADC or MN20A10-ADC by xCELLigence assay

[0384] 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), the assay was performed on an xCELLigence RTCA MP instrument (Agilent). Target cancer cells were seeded onto multi-electrode well plates at a density of 5000 cells / well (100 μL of 50,000 cells / mL stock solution) and incubated in a 37°C / 5% CO2 incubator for 24 hours. mAb MNC2 ADC or mAb MN20A10-ADC was prepared as a 2X solution 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 paclitaxel instead of ADC. Cell killing was evaluated after incubation for 40-120 hours in a 37°C / 5% CO2 incubator. In this assay, impedance was measured in real time. When adherent cancer cells are killed and fall off the electrode surface, the impedance (insulation) decreases.

[0385] The ability of MNC2-ADC and MN20A10-ADC to kill both cells expressing low and high MUC1* was also determined by using the xCELLigence instrument to monitor killing in real time. In the xCelligence system, adherent target cancer cells are coated onto an electrode array 96-well plate. Adherent cells insulate the electrodes and increase impedance. The number of adherent cancer cells is proportional to the impedance. Antibodies and antibody-drug conjugates are much smaller and do not contribute significantly to impedance. Therefore, an increase in impedance reflects the growth of cancer cells, and a decrease in impedance reflects the killing of cancer cells.

[0386] 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 still lower levels of MUC1*. Fig.24 , Fig.26 and Fig. 27 As can be seen in Figure 25, these cancer cells expressing low levels of MUC1* (consistent with early stage cancer cells) were effectively killed by MNC2-ADC and MN20A10-ADC when administered at mid-nanomolar doses. The breast cancer cell lines T47D-MUC1* and HPAFII-MUC1* have been engineered to express high levels of MUC1*, consistent with advanced cancer. Fig.28 As can be seen in the figure, cancer cells expressing high levels of MUC1* are completely killed at very low nanomolar doses. Figure 49A-49D The killing efficacy of MNC2-MMAE, MN20A10-MMAE, MNC2-MMAF and MN20A10-MMAF on T47D breast cancer cells expressing low to moderate levels of MUC1* is shown. As can be seen from the figure, both anti-MUC1* antibodies MNC2 and MN20A10 effectively killed target cells when administered at a concentration of 500 nM over a 40 hour time frame. Figures 49E-49J As can be seen in Figure 2, effective killing was observed at concentrations as low as 167 nM after 120 hours. In fact, MN20A10-MMAE showed effective killing at 56 nM. Comparison here with MNC2-drutecan and MN20A10-drutecan showed comparable killing at the same concentration of 167 nM to MNC2-MMAE, MNC2-MMAF, MN20A10-MMAE, and MN20A10-MMAF.

[0387] In the next set of experiments, the ability of MNC2-ADC and MN20A10-ADC to kill cancer cells that expressed high levels of MUC1* was tested. The expectation was that the higher the levels of MUC1*, the harder it would be to kill the cells, since MUC1* is a growth factor receptor that drives the growth of these cells. Unexpectedly, the experiments showed that the more MUC1* was expressed, the easier it was to kill the cells. Figures 50A-50D It was shown that T47D breast cancer cells engineered to express high levels of MUC1* (indicating advanced cancer) were effectively killed by MNC2-MMAE, MNC2-MMAF, and MN20A10-MMAE at concentrations as low as 19 nM and even as low as 6 nM. As shown in Figures N25E-N25J, when the experiment was allowed to proceed for 120 hours, almost complete killing of cancer cells expressing high levels of MUC1* was seen at concentrations as low as 6 nM for MNC2-MMAE, MNC2-MMAF, and MNC2-Drutecan. For MN20A10, the amount of MMAF and Drutecan that could attach was less, and cancer cell killing was seen at higher concentrations between 19 nM and 167 nM.

[0388] Similar to breast cancer cells expressing low levels of MUC1*, NCI-H1975 non-small cell lung cancer cells expressing even lower levels of MUC1* entered arrest at 40 hours but were killed at 120 hours when treated with MNC2-MMAE, MNC2-MMAF, MNC2-delutec, MN20A10-MMAE, or MN20A10-MMAF ( Figure 51A-Figure 51I HPAF II-wt pancreatic cancer cells, which expressed MUC1* at a lower level but higher than H1975 lung cancer cells, were killed by MNC2-MMAE, MNC2-MMAF, MN20A10-MMAE, and MN20A10-MMAF at concentrations ranging from 167 nM to 500 nM at 40 h, and the killing rate increased to 56 nM after 120 h ( Figure 52A-Figure 52I ). However, if pancreatic cells were engineered to express high levels of MUC1*, MNC2-ADC and MN20A10-ADC were more effective in killing cancer cells. After about 40 hours, almost complete killing of cancer cells was seen at concentrations between 167nM and 56nM, while after 120 hours, almost complete killing of cancer cells was achieved at concentrations between 19nM and 56nM ( Figure 53A-Figure 53I ).

[0389] The toxin exatecan was then conjugated to MNC2 via a linker comprising a maleimidocaproyl group, a valine-citrulline group, and a p-aminobenzyl group, as shown in Figures N4E-N4H. In addition, the conjugation was performed in a solution containing propylene glycol MES pH 6.0 buffer.

[0390] like Figures 65A-65D As shown, tumor cells implanted in animals show a significant increase in MUC1* expression as the tumor progresses 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 lines provided by ATCC have been expanded thousands or even millions of times. Essentially all T47D cells are identical. Staining of cells at day 0 showed that the cells expressed more full-length MUC1 than MUC1*, and the staining intensity was lighter, indicating a low number of MUC1* receptors, which indicates an early stage cancer. Sixty-two (62) days after tumor implantation, which is equivalent to approximately 7 years in human time, it can be easily seen that MUC1* expression (in terms of expression level and expression intensity) has significantly changed from low expression to high expression in late stage tumors. In contrast, staining of serial sections of the tumor showed expression of full-length MUC1 as expected because it is cleaved into MUC1* after surface expression. However, the staining intensity did not increase, indicating that the vast majority of MUC1 expressed in late stage tumors has been cleaved into the growth factor receptor form MUC1*.

[0391] As cancer clinicians and pathologists know, tumors that form in patients are not homogeneous populations of a single clone like cell lines. Actual tumors are heterogeneous to some extent in that they consist of both low and high antigen expressing cells. However, it is also well known that early stage cancers are characterized by the majority of cells being low expressing cells, whereas late stage cancers are characterized by the majority of cells being high expressing cells.

[0392] Example 17 Measurement of target cell killing by MNC2-ADC or MN20A10-ADC by PrestoBlue live / dead cell assay

[0393] Another method to measure the killing activity of mAb MNC2-ADC and mAb MN20A10-ADC on target cancer cells (including T47D breast cancer cells, NCI-H1975 non-small cell lung cancer cells, and HPAFII pancreatic cancer cells) is to use the PrestoBlue live / dead cell assay (ThermoFisher). The target cancer cells were seeded in a black-walled, clear-bottomed 96-well tissue culture plate at a density of 5000 cells / well (100 μL of 50,000 cells / mL stock solution) 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), cancer cells were treated with 1 μM paclitaxel instead of ADC. After incubation in a 37°C / 5% CO2 incubator for 72 hours-120 hours, cell viability was measured by fluorescence using the PrestoBlue HS assay (ThermoFisher). In brief, after the incubation period, 20 μL of PrestoBlue HS solution was added. After incubation for 30-90 min, fluorescence (Ex 560 nm / Em 590 nm) was recorded using a Tecan plate reader. Viability percentages were determined by PrestoBlue HS staining and normalized to cells treated with 1 μM paclitaxel.

[0394] The normalized fluorescence values ​​were input into the graphing program Sigma Plot, and the data were plotted as the relationship between the MNC2-ADC or MN20A10-ADC concentration and the normalized fluorescence signal, and the curve was fitted using the four-parameter Hill formula: f = y0 + a*x^b / (c^b + x^b)

[0395] IC50 values ​​were calculated from the fitting data of the killing ability of MNC2-ADC and MN20A10-ADC against target cancer cells and reported in nM.

[0396] 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 HPAFII pancreatic cancer cells) were taken on an Olympus IX-71 microscope at 4x or 20x magnification after 120 h of incubation with the indicated MNC2-ADC or MN20A10-ADC concentrations.

[0397] Figure 46A-Figure 48NMagnified photographs of various cancer cell types after treatment with MNC2-ADC or MN20A10-ADC are shown. Killing can be easily seen as a significant reduction in cell number, a change in cell morphology from a characteristic flat, spread morphology to a rounded morphology and cell shedding. In contrast, control wells showed a confluent monolayer of dense cells with a normal flat spread morphology and no dead floating cells. Figure 46A-46C and Figure 46G-Figure 46F Shown are T47D-MUC1* breast cancer cells treated with MNC2-MMAE. Figure 46D-Figure 46F and Figure 46G-Figure 46J Shown are T47D-MUC1* breast cancer cells treated with MNC2-derutec. Figure 47A-47B Shown are DU145 hormone-resistant prostate cancer cells treated with MNC2-MMAE. Figure 47C-Figure 47D Shown are DU145 hormone-resistant prostate cancer cells treated with MNC2-derutecan. Figure 48A-48B and Figure 48G-Figure 48H Shown are T47D-MUC1* breast cancer cells treated with MN20A10-MMAE. Figure 48C-Figure 48D and Figure 48I-48J Shown are T47D-MUC1* breast cancer cells treated with MN20A10-deruteccan. Figure 48K-48L Shown are DU145 hormone-resistant prostate cancer cells treated with MN20A10-MMAE. Figure 48M-48N DU145 hormone-resistant prostate cancer cells treated with MN20A10-drutecan are shown. Figure 46A-Figure 48N The killing effect of anti-MUC1*-ADC shown in Figures 38A-45D The killing was consistent with the flow cytometry measurements shown in .

[0398] Example 18. Animal studies: in vivo measurement of target cell killing by MNC2-ADC or MN20A10-ADC

[0399] In vivo experiments were performed to measure the killing of xenograft target cancer cells by MNC2-ADC or MN20A10-ADC.

[0400] For breast cancer xenografts, female NOD / SCID mice (18-22 g body weight, 6-8 weeks old, Charles River Laboratories) previously implanted with a 90-day estrogen-releasing pellet (Innovative Research Laboratories) were injected subcutaneously with one million luciferase-mCherry-positive T47D breast cancer cells expressing moderate to high levels of MUC1* (ATCC) under isoflurane anesthesia (ISOTHESIA, 250 ml (HENRY SCHEN, NDC: 11695-6776-2)) using a BD 26G insulin syringe with a detachable needle (Fisher Catalog No.: 329652) or a BD 28G Lo-Dose U-100 insulin syringe (Fisher Catalog No.: 329461).

[0401] For lung cancer xenografts or pancreatic cancer xenografts, female NU / NU mice (18-22 g, 6-8 weeks old, Charles River Laboratories) were subcutaneously injected with one million luciferase-mCherry-positive NCI-H1975 non-small cell lung cancer cells (ATCC) or one million luciferase-mCherry-positive HPAFII pancreatic cancer cells (ATCC), which express low to moderate levels of MUC1*, under isoflurane anesthesia (ISOTHESIA, 250 ml (HENRY SCHEN, NDC: 11695-6776-2)).

[0402] 4-6 days after xenografting, mice were injected intraperitoneally with 150ul, 30mg / mL D-luciferin (XenoLight TM D-luciferin potassium salt; PerkinElmer P / N 122799) was placed into the scruff of the neck and then placed under isoflurane anesthesia before bioluminescent image acquisition using the XenogenIVIS-Spectrum system (Perkin Elmer). Group selection was performed to evenly distribute xenograft mice to ensure that the mock-treated and ADC-treated groups had the same initial tumor size.

[0403] On days 5-7 after xenografting, mice were injected intraperitoneally with phosphate buffered saline (PBS) or 5-10 mg / kg MNC2-ADC or 5-10 mg / kg MN20A10-ADC. Thereafter, mice were injected once a week for a total of four injections of MNC2-ADC or MN20A10-ADC.

[0404] Bioluminescence imaging was performed weekly to non-invasively measure tumor growth in real time. Data are presented graphically as radiance (photons / sec) as a function of days post tumor implantation.

[0405] For some tumor growth measurements, caliper measurements were performed on the tumors of 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 be equal to the shortest of the perpendicular axes, defined as y. Measurements were performed with a digital vernier caliper while the mouse was awake and calculated according to the following formula: Xenograft volume = xy 2 / 2.

[0406] Humane criteria for euthanasia were followed, including: tumor burden reaching or exceeding 20 mm in diameter (in accordance with IACUC policy), tumor ulceration, or tumor location interfering with normal ambulation, eating / drinking, and / or elimination; body condition score of 2 or weight loss >15% (compared to pre-xenotransplantation weight).

[0407] In vivo experiments were also performed to evaluate the killing efficacy of MNC2-ADC and MN20A10-ADC in immunocompromised mice implanted with several different types of human cancers, some of which expressed low levels of MUC1* and others that expressed high levels of MUC1*. In all cases, the human cancer cell lines had been engineered to express mCherry and luciferase so that tumors could be measured by bioluminescence. To optically measure tumor volume, the luciferase substrate luciferin was injected into the animals and bioluminescence was photographed on an IVIS instrument within 10 minutes of luciferin injection. The expression levels of luciferase varied, and in some cases greatly, making it challenging to compare tumor size between different cell lines. In these cases, caliper measurements of the tumors were also taken to allow comparison of tumor size between cell lines. Additionally, in these cases, photographs of the animals were taken so that tumor size between cell lines could be visually compared.

[0408] In one experiment, MNC2-MMAE was administered to female NOD / SCID / GAMMA (NSG) mice carrying a 90-day estrogen-releasing pellet and then implanted with either 1M T47D-wt breast cancer cells, which express low to moderate levels of MUC1*, indicating early-stage cancer, or 1M T47D-MUC1* cells, which were engineered to express high levels of MUC1*, indicating advanced cancer. Tumors were allowed to implant for seven (7) days prior to the first injection of MNC2-MMAE at a dose of 5 mg / kg. The dose was increased to 10 mg / kg on days 14 and 20. First, mice were implanted with T47D-wt tumors, which express low levels of MUC1*. Fig.57AControl mice and Fig.57B By comparing the MNC2-MMAE treated mice in the experiment to the MNC2-MMAE treated mice in the experiment, it can be easily seen that the treated mice survived with very small tumor burdens until the end of the experiment on day 56. In contrast, the tumors of the untreated control mice continued to grow until they had to be sacrificed on day 56 due to excessive tumor burden. Next, mice implanted with T47D-MUC1* tumors expressing high levels of MUC1* were examined. Control animals implanted with T47D-wt ( Fig.57A and Fig.57E ) and animals implanted with T47D-MUC1* ( Fig.57C and Fig.57F ) compared to the bioluminescence of the T47D-MUC1* cell line, the T47D-MUC1* cell line expressed slightly less luciferase than the T47D-wt cell line. However, visual inspection of the tumors showed that tumors of comparable size were present in both cell lines. Animals bearing T47D-MUC1* tumors were administered MNC2-MMAE on the same schedule as animals bearing T47D-wt tumors. However, Fig.57D It can be clearly seen that the animals treated with MNC2-MMAE were tumor-free from day 34 until the end of the experiment on day 56, when the experiment was terminated because the control mice became overloaded with tumors and had to be sacrificed. The same experiment was performed in parallel, but the animals were given MN20A10-MMAE ( Figures 58A-58F ). The MN20A10-MMAE results followed the same trend as the MNC2-MMAE results, but MNC2-MMAE was more potent than MN20A10. A possible explanation for this difference is the different number of toxins attached to the MNC2 antibody (DAR 3.85) and MN20A10 (DAR 2.96).

[0409] In another experiment, female nu / nu mice were implanted with 1M human NCI-H1975 non-small cell lung cancer cells that express low to moderate levels of MUC1*, indicating early-stage cancer. The animals were injected with 5mg / kg of MNC2-MMAE on days 7 and 14 after tumor implantation. On day 19, the dose was increased to 10mg / kg. The bioluminescent photos clearly showed that increasing the dose of MNC2-MMAE to 10mg / kg on days 19 and 27 increased the killing of tumor cells ( Figure 59A-Figure 59C). The efficacy of MNC2-MMAE treatment can be understood by comparing the tumor weights of animals sacrificed in the control and treatment groups. Control animals had to be sacrificed on day 20 post-implantation due to life-threatening tumor sizes, with tumor weights of 0.82, 0.3, 0.49, 0.41, and 0.5 grams. In contrast, animals in the treatment group weighed 0.28 and 0.4 grams that needed to be sacrificed on day 54. Two mice in the treatment group were still tumor-free on day 67, at which time the experiment was arbitrarily terminated. Results from parallel experiments showed the same trend, in which animals were treated with MN20A10-MMAE ( Figures 60A-60D ), although the effect of MN20A10-MMAE was less pronounced than that of MNC2-MMAE, probably because of the lower DAR of MN20A10-MMAE. However, treatment suppressed tumor growth, as can be seen from the bioluminescence photos and from the weight of tumors removed from control animals that had to be sacrificed on day 26. Tumor weights ranged from 2.2 g to 0.65 g. Fig.60D The graph shows a dramatic decrease in tumor volume after the dose was increased to 10 mg / kg on day 19. The data suggest that tumor growth would have been more significantly reduced if the dose had been higher initially.

[0410] In another experiment, MNC2-MMAE was administered to female nu / nu (NSG) mice implanted with either 0.5M HPAF II-wt pancreatic cancer cells, which express low to moderate levels of MUC1*, indicative of early stage cancer, or 0.5M HPAF II-MUC1* pancreatic cancer cells, which are engineered to express high levels of MUC1*, indicative of advanced stage cancer. Tumors were allowed to implant for five (5) days, then three (3) injections of MNC2-MMAE were given at a dose of 10 mg / kg on days 5, 12, and 19. First, mice implanted with HPAF II-wt tumors, which express low levels of MUC1*, were tested. Fig.61A Control mice and Fig.61B By comparing the MNC2-MMAE treated mice in the 18-well plate, it can be easily seen that the tumors in the treated mice were smaller than those in the control mice at day 18. The IVIS instrument malfunctioned at day 25, causing artificially low readings in the control animals. For this reason, caliper measurements were also taken at day 25. Fig.61H As can be seen, the mean tumor volume measured by caliper differs greatly between control animals and treated animals implanted with HPAF II-wt tumors.

[0411] Next, mice implanted with T47D-MUC1* tumors expressing high levels of MUC1* were tested. Fig.57A and Fig.57E) and animals implanted with T47D-MUC1* ( Fig.57C and Fig.57F ) bioluminescence, the T47D-MUC1* line expressed slightly less luciferase than the T47D-wt cell line. However, visual inspection of the tumors showed comparable tumor size between the two cell lines. Animals bearing T47D-MUC1* tumors were administered MNC2-MMAE on the same schedule as animals bearing T47D-wt tumors. However, viewing Fig.57D It can be clearly seen that the MNC2-MMAE treated animals were tumor free from day 34 until the end of the experiment on day 56, when the experiment was terminated because the control mice became overloaded with tumors and had to be sacrificed.

[0412] Implementation

[0413] Embodiment 1. A conjugate of formula (I): [Ab]-[ZLRX] y Formula (I), 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 coupling moiety; L is a dipeptide, tripeptide or tetrapeptide linking moiety having Z bound to the N-terminus and R bound to the C-terminus; [Ab] is an antibody comprising an anti-MUC1* binding domain, the 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 R2 and MUC1*HC-CDR3 comprise an amino acid sequence selected from those specified in Table 1; wherein 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, MUC1*LC-CDR2 and MUC1*LC-CDR3 of the MUC1* binding domain comprise an amino acid sequence selected from those specified in Table 1; Z is a conjugated moiety capable of forming a covalent bond with the sulfur atom of a cysteine ​​residue; and wherein y is an integer from 1 to 10.

[0414] Embodiment 2. The conjugate of embodiment 1, wherein L comprises valine and citrulline.

[0415] Embodiment 3. The conjugate of embodiment 1, wherein L comprises glycine and phenylalanine.

[0416] Embodiment 4. The conjugate of embodiment 1, wherein R comprises p-aminobenzyl.

[0417] Embodiment 5. The conjugate of embodiment 1, wherein R comprises the structure where * indicates the point of attachment of the X group.

[0418] Embodiment 6. The conjugate of embodiment 1, wherein R comprises the structure where * indicates the point of attachment of the X group.

[0419] Embodiment 7. The conjugate of embodiment 1, wherein R comprises the structure where * indicates the attachment point of the X group

[0420] Embodiment 8. The conjugate of embodiment 1, wherein L is a compound comprising the structure The dipeptide linking portion.

[0421] Embodiment 9. The conjugate of embodiment 1, wherein L is a compound comprising the structure The tetrapeptide linker portion.

[0422] Embodiment 10. The conjugate of embodiment 1, wherein X is MMAE or MMAF.

[0423] Embodiment 11. The conjugate of embodiment 1, wherein X is exatecan or Dxd

[0424] Embodiment 12. The conjugate of embodiment 1, wherein R comprises wherein * represents the point of attachment of the X group, and wherein X is Dxd.

[0425] Embodiment 13. The conjugate of embodiment 1, wherein R comprises the structure wherein * represents the point of attachment of the X group, and wherein X is exatecan.

[0426] Embodiment 14. The conjugate of embodiment 1, wherein the antibody is of isotype IgG1 or IgG2.

[0427] Embodiment 15. The conjugate of embodiment 1, wherein the antibody is of isotype IgG2b.

[0428] 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.

[0429] Embodiment 17. The conjugate of 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.

[0430] 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.

[0431] Embodiment 19. The conjugate of 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.

[0432] Embodiment 20. The conjugate of 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.

[0433] 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, wherein the payload comprises a linker and a cytotoxic compound, wherein the cytotoxic compound comprises a tubulin inhibitor or a topoisomerase I inhibitor.

[0434] Embodiment 22. The antibody conjugate of Embodiment 21, wherein the linker comprises valine.

[0435] Embodiment 23. The antibody conjugate of Embodiment 21, wherein the linker comprises citrulline.

[0436] Embodiment 24. The antibody conjugate of embodiment 21, wherein the linker comprises valine and citrulline.

[0437] Embodiment 25. The antibody conjugate of embodiment 21, wherein the linker comprises the structure The dipeptide linking portion.

[0438] Embodiment 26. The antibody conjugate of embodiment 21, wherein the linker comprises at least one glycine.

[0439] Embodiment 27. The antibody conjugate of embodiment 21, wherein the linker comprises at least one glycine and a phenylalanine.

[0440] Embodiment 28. The antibody conjugate of embodiment 21, wherein the linker comprises the structure

[0441] Embodiment 29. The antibody conjugate of Embodiment 21, wherein the linker comprises a p-aminobenzyl group.

[0442] Embodiment 30. The antibody conjugate of embodiment 21, wherein the linker comprises a structural group where * indicates the attachment point of the cytotoxic group.

[0443] Embodiment 31. The antibody conjugate of embodiment 21, wherein the linker comprises a structural group where * indicates the attachment point of the cytotoxic group.

[0444] Embodiment 32. The antibody conjugate of embodiment 21, wherein the tubulin inhibitor is MMAE or MMAF.

[0445] Embodiment 33. The antibody conjugate of embodiment 21, wherein the topoisomerase I inhibitor is exatecan or derutecan, or a derivative thereof.

[0446] Embodiment 34. The antibody conjugate of embodiment 21, wherein the linker comprises a structure wherein * indicates the point of attachment of the cytotoxic group and wherein the topoisomerase I inhibitor is Dxd.

[0447] Embodiment 35. The antibody conjugate of embodiment 21, wherein the linker comprises a structure wherein * indicates the point of attachment of the cytotoxic group, and wherein the topoisomerase I inhibitor is exatecan.

[0448] Embodiment 36. The antibody conjugate of Embodiment 21, comprising the structure provided below, wherein n is 1 to 10:

[0449] Embodiment 37. The antibody conjugate of Embodiment 21, comprising the structure provided below, wherein n is 1 to 10:

[0450] Embodiment 38. The antibody conjugate of embodiment 21, wherein the antibody isotype is IgG1 or IgG2.

[0451] Embodiment 39. The antibody conjugate of Embodiment 21, wherein the antibody isotype is IgG2b.

[0452] Embodiment 40. The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least two payloads.

[0453] Embodiment 41. The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least three payloads.

[0454] Embodiment 42. The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least four payloads.

[0455] Embodiment 43. The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least five payloads.

[0456] Embodiment 44. The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least six payloads.

[0457] Embodiment 45. The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least seven payloads.

[0458] Embodiment 46. The antibody conjugate of embodiment 21, wherein the antibody is conjugated to at least eight payloads.

[0459] 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; wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the MUC1* binding domain comprise an amino acid sequence selected from those specified in Table 1; and wherein at least one of LC-CDR1, LC-CDR2, and LC-CDR3 comprises 0-2 amino acid modifications.

[0460] 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; wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the MUC1* binding domain comprise an amino acid sequence selected from those specified in Table 1; and wherein at least one of HC-CDR1, HC-CDR2, and HC-CDR3 comprises 0-2 amino acid modifications.

[0461] Embodiment 49. The antibody conjugate of Embodiment 21, wherein the anti-MUC1* binding domain comprises a heavy chain variable domain comprising a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences set forth in Table 2.

[0462] Embodiment 50. The antibody conjugate of Embodiment 21, wherein the anti-MUC1* binding domain comprises a light chain variable domain comprising a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences set forth in Table 2.

[0463] Embodiment 51. The antibody conjugate of Embodiment 21, wherein the anti-MUC1* binding domain comprises a single chain variable fragment (scFv) comprising a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence specified in Table 3.

[0464] Embodiment 52. An antibody comprising a MUC1* binding domain and a CD3 binding domain, wherein the MUC1* binding domain comprises three heavy chain (HC) complementary 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; wherein the MUC1* binding domain comprises three light chain (LC) complementary 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, MUC1* LC-CDR2 comprises the amino acid sequence of SEQ ID NO: 14, and MUC1* LC-CDR3 comprises the amino acid sequence of SEQ ID NO: 3. NO:15; wherein the CD3 binding domain comprises 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 the group consisting of those specified 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 CD3LC-CDR3; wherein CD3 LC-CDR1, CD3 LC-CDR2 and CD3 LC-CDR3 of the CD3 binding domain comprise an amino acid sequence selected from the group consisting of those specified in Table 4.

[0465] Embodiment 53. The antibody of Embodiment 52, wherein the antibody comprises an Fc domain.

[0466] Embodiment 54. The antibody of Embodiment 52, wherein the Fc domain is a heterodimeric Fc domain.

[0467] Embodiment 55. The antibody of embodiment 52, wherein the heterodimeric Fc domain comprises a knob chain and a hole chain, forming a knob-hole (KiH) structure.

[0468] Embodiment 56. The antibody of Embodiment 55, wherein the knob chain comprises a sequence that is at least about 95% identical to a sequence selected from SEQ ID NO: 121, 122, 123, or 124.

[0469] Embodiment 57. The antibody of Embodiment 55, wherein the holmium chain comprises a sequence that is at least about 95% identical to a sequence selected from SEQ ID NO: 125, 126, 127 or 128.

[0470] Embodiment 58. The antibody of Embodiment 52, wherein the MUCl* binding domain comprises a heavy chain variable domain comprising a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the amino acid sequences of Table 2.

[0471] Embodiment 59. The antibody of Embodiment 52, wherein the MUCl* binding domain comprises a light chain variable domain comprising a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from Table 2.

[0472] Embodiment 60. The antibody of Embodiment 52, wherein the MUCl* 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.

[0473] Embodiment 61. The antibody of Embodiment 52, wherein the CD3 binding domain comprises a heavy chain comprising a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 26 or 31.

[0474] Embodiment 62. The antibody of Embodiment 52, wherein the CD3 binding domain comprises a light chain comprising a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 29 or 35.

[0475] 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.

[0476] 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 NO: 50, 52, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110 or 114.

[0477] Embodiment 65. A method of treating cancer, comprising administering the antibody or antibody conjugate of any one of Embodiments 1-64 to a subject in need thereof.

[0478] Embodiment 66. The method of Embodiment 65, wherein the cancer expresses MUCl*.

[0479] Embodiment 67. The method of Embodiment 65, wherein the cancer is breast cancer, colon cancer, prostate cancer, pancreatic cancer, or lung cancer.

Claims

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

2. The conjugate according to claim 1, wherein L comprises valine and citrulline.

3. The conjugate of claim 1, wherein L comprises glycine and phenylalanine. The conjugate according to claim 1 , wherein R comprises p-aminobenzyl.

5. The conjugate according to claim 1, wherein R comprises the structure wherein * indicates the point of attachment of the X group.

6. The conjugate according to claim 1, wherein R comprises the structure wherein * indicates the point of attachment of the X group.

7. The conjugate according to claim 1, wherein R comprises the structure wherein * indicates the point of attachment of the X group.

8. The conjugate according to claim 1, wherein L is a The dipeptide linking portion.

9. The conjugate according to claim 1, wherein L is a The tetrapeptide linker portion.

10. The conjugate of claim 1, wherein X is MMAE or MMAF.

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

12. The conjugate according to claim 1, wherein R comprises the structure wherein * represents the point of attachment of the X group, and wherein X is Dxd.

13. The conjugate according to claim 1, wherein R comprises the structure wherein * represents the point of attachment of the X group, and wherein X is exatecan. The conjugate of claim 1 , wherein the antibody is of isotype IgG1 or IgG2.

15. The conjugate of claim 1, wherein the antibody is of isotype IgG2b.

16. The conjugate of claim 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.

17. The conjugate of claim 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.

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

19. The conjugate of claim 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.

20. The conjugate of claim 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.

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, wherein the payload comprises a linker and a cytotoxic compound, wherein the cytotoxic compound comprises a tubulin inhibitor or a topoisomerase I inhibitor.

22. The antibody conjugate of claim 21, wherein the linker comprises valine.

23. The antibody conjugate of claim 21, wherein the linker comprises citrulline.

24. The antibody conjugate of claim 21, wherein the linker comprises valine and citrulline.

25. The antibody conjugate of claim 21, wherein the linker comprises the structure The dipeptide linking portion.

26. The antibody conjugate of claim 21, wherein the linker comprises at least one glycine.

27. The antibody conjugate of claim 21, wherein the linker comprises at least one glycine and phenylalanine.

28. The antibody conjugate of claim 21, wherein the linker comprises the structure 29. The antibody conjugate of claim 21, wherein the linker comprises a p-aminobenzyl group.

30. The antibody conjugate of claim 21, wherein the linker comprises a structural group Wherein * indicates the attachment point of the cytotoxic group.

31. The antibody conjugate of claim 21, wherein the linker comprises a structural group Wherein * indicates the attachment point of the cytotoxic group.

32. The antibody conjugate of claim 21, wherein the tubulin inhibitor is MMAE or MMAF.

33. The antibody conjugate of claim 21, wherein the topoisomerase I inhibitor is exatecan or derutecan, or a derivative thereof.

34. The antibody conjugate of claim 21, wherein the linker comprises a structure A group wherein * represents the point of attachment of the cytotoxic group, and wherein the topoisomerase I inhibitor is Dxd.

35. The antibody conjugate of claim 21, wherein the linker comprises a structure wherein * represents the point of attachment of the cytotoxic group, and wherein the topoisomerase I inhibitor is exatecan.

36. The antibody conjugate of claim 21, comprising the structure provided below, wherein n is 1 to 10:

37. The antibody conjugate of claim 21, comprising the structure provided below, wherein n is 1 to 10:

38. The antibody conjugate of claim 21, wherein the antibody isotype is IgG1 or IgG2.

39. The antibody conjugate of claim 21, wherein the antibody isotype is IgG2b.

40. The antibody conjugate of claim 21, wherein the antibody is conjugated to at least two payloads.

41. The antibody conjugate of claim 21, wherein the antibody is conjugated to at least three payloads.

42. The antibody conjugate of claim 21, wherein the antibody is conjugated to at least four payloads.

43. The antibody conjugate of claim 21, wherein the antibody is conjugated to at least five payloads.

44. The antibody conjugate of claim 21, wherein the antibody is conjugated to at least six payloads.

45. The antibody conjugate of claim 21, wherein the antibody is conjugated to at least seven payloads.

46. ​​The antibody conjugate of claim 21, wherein the antibody is conjugated to at least eight payloads.

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

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

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

50. The antibody conjugate of claim 21, wherein the anti-MUC1* binding domain comprises a light chain variable domain comprising a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in Table 2.

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

52. An antibody comprising a MUC1* binding domain and a CD3 binding domain, wherein the MUC1* binding domain comprises three heavy chain (HC) complementarity determining regions (CDRs): MUC1* HC-CDR1, MUC1* HC-CDR2, and MUC1* HC-CDR3; wherein the MUC1* HC-CDR1 comprises the amino acid sequence of SEQ ID NO: 1, the MUC1* HC-CDR2 comprises the amino acid sequence of SEQ ID NO: 2, and the MUC1* HC-CDR3 comprises the amino acid sequence of SEQ ID NO: 3; wherein the MUC1* binding domain comprises three light chain (LC) complementarity determining regions (CDRs): MUC1*LC-CDR1, MUC1*LC-CDR2, and MUC1*LC-CDR3; wherein the MUC1*LC-CDR1 comprises the amino acid sequence of SEQ ID NO: 13, the MUC1*LC-CDR2 comprises the amino acid sequence of SEQ ID NO: 14, and the MUC1*LC-CDR3 comprises the amino acid sequence of SEQ ID NO: 15; in, The CD3 binding domain comprises three heavy chain (HC) complementarity determining regions (CDRs): CD3 HC-CDR1, CD3 HC-CDR2 and CD3 HC-CDR3; wherein the CD3 HC-CDR1, the CD3 HC-CDR2 and the CD3 HC-CDR3 of the CD3 binding domain comprise an amino acid sequence selected from the group consisting of those specified in Table 2; and in, The CD3 binding domain comprises three light chain (LC) complementarity determining regions (CDRs): CD3 LC-CDR1, CD3 LC-CDR2 and CD3 LC-CDR3; wherein the CD3 LC-CDR1, the CD3 LC-CDR2 and the CD3 LC-CDR3 of the CD3 binding domain comprise an amino acid sequence selected from the group consisting of those specified in Table 4.

53. The antibody of claim 52, wherein the antibody comprises an Fc domain.

54. The antibody of claim 52, wherein the Fc domain is a heterodimeric Fc domain.

55. The antibody of claim 52, wherein the heterodimeric Fc domain comprises a knob chain and a hole chain, forming a knob-hole (KiH) structure.

56. The antibody of claim 55, wherein the knob chain comprises a sequence that is at least about 95% identical to a sequence selected from SEQ ID NO: 121, 122, 123, or 124.

57. The antibody of claim 55, wherein the holmium chain comprises a sequence that is at least about 95% identical to a sequence selected from SEQ ID NO: 125, 126, 127 or 128.

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

59. The antibody of claim 52, wherein the MUCl* binding domain comprises a light chain variable domain comprising a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from Table 2.

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

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

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

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

64. The antibody of claim 52, comprising a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical 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.

65. A method of treating cancer, comprising administering to a subject in need thereof the antibody or antibody conjugate of any one of claims 1-64.

66. The method of claim 65, wherein the cancer expresses MUCl*.

67. The method of claim 65, wherein the cancer is breast cancer, colon cancer, prostate cancer, pancreatic cancer, or lung cancer.

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