First cell-targeting bispecific molecules

Abstract

The subject matter described herein relates to methods of treating cancer in a subject in need thereof using a bispecific molecule that recognizes two antigenic markers of different tissue lineages on the surface of a primary cell.

Description

[Technical field]

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 295,681, filed December 31, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile by anyone of the patent document or the patent disclosure as set forth in this application, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all and any copyright rights whatsoever.

[0003] All cited patents, patent applications, and publications are incorporated herein by reference in their entireties, and the disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art known to those skilled in the art herein as of the date of the inventions described herein. [Background technology]

[0004] Cancer refers to a group of conditions characterized by abnormal cell growth. Cancer cells can spread and invade other organs in the body. The most common symptoms of cancer include lumps, abnormal bleeding, persistent coughing, and unexplained weight loss. There are also blood cancers that do not form cell masses. More than 100 types of cancer can occur in the human body, and most of them are incurable. Summary of the Invention

[0005] In certain aspects, the subject matter described herein provides bispecific molecules comprising at least two antigen-binding regions, each antigen-binding region binding to a different antigen on the cancer's first cell (TFC).

[0006] In some embodiments, the first antigen is an epithelial cell lineage marker. In some embodiments, the epithelial cell lineage marker is any one of the markers in FIG. 2. In some embodiments, the epithelial cell lineage marker is epithelial cell adhesion molecule (EpCAM). In some embodiments, EpCAM comprises SEQ ID NO:7 or SEQ ID NO:12. In some embodiments, the second antigen is a macrophage cell lineage marker. In some embodiments, the macrophage cell lineage marker is any one of the markers in FIG. 1. In some embodiments, the macrophage cell lineage marker is CD163. In some embodiments, CD163 comprises SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:13.

[0007] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is breast cancer, brain cancer, gastrointestinal cancer, pancreatic cancer, renal cancer, liver cancer, lung cancer, thymus cancer, ovarian cancer, prostate cancer, or endometrial cancer. In some embodiments, the gastrointestinal cancer is gastric cancer or colon cancer.

[0008] In some embodiments, the cancer comprises a liquid cancer. In some embodiments, the liquid cancer is a leukemia, lymphoma, or myeloma. In some embodiments, the liquid cancer is acute myeloid leukemia (AML). In some embodiments, the liquid cancer is a B-cell malignancy. In some embodiments, the liquid cancer is a myeloid neoplasm, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS / MPN overlap syndrome, acute myeloid leukemia, or chronic myeloid leukemia.

[0009] In some embodiments, the first antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 36, a light chain CDR2 (CDRL2) of SEQ ID NO: 37, and a light chain CDR3 (CDRL3) of SEQ ID NO: 38, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 33, a heavy chain CDR2 of SEQ ID NO: 34, and a heavy chain CDR3 of SEQ ID NO: 35. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 42, a light chain CDR2 (CDRL2) of SEQ ID NO: 43, and a light chain CDR3 (CDRL3) of SEQ ID NO: 44, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 39, a heavy chain CDR2 of SEQ ID NO: 40, and a heavy chain CDR3 of SEQ ID NO: 41. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 48, a light chain CDR2 (CDRL2) of SEQ ID NO: 49, and a light chain CDR3 (CDRL3) of SEQ ID NO: 50, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 45, a heavy chain CDR2 of SEQ ID NO: 46, and a heavy chain CDR3 of SEQ ID NO: 47.

[0010] In some embodiments, the first antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 28 and a heavy chain variable (VH) region of SEQ ID NO: 27. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 30 and a heavy chain variable (VH) region of SEQ ID NO: 29. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 32 and a heavy chain variable (VH) region of SEQ ID NO: 31.

[0011] In some embodiments, the first antigen and the second antigen are macrophage cell lineage markers. In some embodiments, the first antigen is CD117, CD34, or CD123, and the second antigen is CD163. In some embodiments, the TFC is a metastatic TFC.

[0012] In some embodiments, the bispecific molecule is a bispecific antibody or an antigen-binding fragment thereof. In some embodiments, the bispecific antibody is conjugated to a drug. In some embodiments, the drug is a toxin. In some embodiments, the drug is a chemotherapeutic agent. In some embodiments, the bispecific molecule comprises a dual nanobody, a BiTE, a tandAb, a DART, a DART-Fc, a DARPin, a scFv, a scFv-HAS-scFV, and a DNL-Fab3. In some embodiments, the bispecific molecule is a bispecific chimeric antigen receptor (CAR). In some embodiments, the bispecific molecule is a Co-LOCKR comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer.

[0013] In some embodiments, the first polypeptide comprises SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 19. In some embodiments, the second polypeptide comprises SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO:21.

[0014] In some embodiments, the bispecific CAR is a synNotch CAR. In some embodiments, the bispecific molecule is Co-LOCKR comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer, and the third polypeptide binds to the CAR, and the third polypeptide is operably linked to the first polypeptide or the second polypeptide.

[0015] In some embodiments, the bispecific molecule comprises a split CAR-T system comprising a chimeric antigen receptor (CAR) module and a chimeric costimulatory receptor (CCR) module, wherein the CAR module comprises a polypeptide comprising a first antigen binding region and a CD3z signaling domain, and the CCR module comprises a polypeptide comprising a second antigen binding region and two or more costimulatory domains, and wherein the CAR module and the CCR module each bind to a different antigen on the TFC of the cancer. In some embodiments, the split CAR-T system comprises one or more of the polypeptide sequences in Table 4.

[0016] In certain aspects, the subject matter described herein provides a pharmaceutical composition comprising a bispecific molecule according to any embodiment described herein.

[0017] In certain aspects, the subject matter described herein provides a polynucleotide encoding a bispecific molecule according to any embodiment described herein.

[0018] In certain aspects, the subject matter described herein provides a vector comprising a polynucleotide according to any embodiment described herein.

[0019] In certain aspects, the subject matter described herein provides a virus comprising a polynucleotide according to any embodiment described herein.

[0020] In certain aspects, the subject matter described herein provides a genetically modified cell comprising a bispecific molecule according to any embodiment described herein.

[0021] In certain aspects, the subject matter described herein provides a genetically modified cell comprising a polynucleotide according to any embodiment described herein.

[0022] In certain aspects, the subject matter described herein provides a method of treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject a bispecific molecule comprising at least two antigen binding regions, each antigen binding region binding to a different antigen on a cancer cell of origin (TFC).

[0023] In some embodiments, the first antigen is an epithelial cell lineage marker. In some embodiments, the epithelial cell lineage marker is any one of the markers in Figure 2. In some embodiments, the epithelial cell lineage marker is an epithelial cell adhesion molecule (EpCAM). In some embodiments, EpCAM comprises SEQ ID NO:7 or SEQ ID NO:12.

[0024] In some embodiments, the second antigen is a macrophage cell lineage marker. In some embodiments, the macrophage cell lineage marker is any one of the markers in Figure 1. In some embodiments, the macrophage cell lineage marker is CD163. In some embodiments, CD163 comprises SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:13.

[0025] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is breast cancer, brain cancer, gastrointestinal cancer, pancreatic cancer, renal cancer, liver cancer, lung cancer, thymic cancer, ovarian cancer, prostate cancer, or endometrial cancer. In some embodiments, the gastrointestinal cancer is gastric cancer or colon cancer.

[0026] In some embodiments, the cancer is a liquid cancer. In some embodiments, the liquid cancer is a leukemia, lymphoma, or myeloma. In some embodiments, the liquid cancer is acute myeloid leukemia (AML). In some embodiments, the liquid cancer is a B-cell malignancy. In some embodiments, the liquid cancer is a myeloid neoplasm, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS / MPN overlap syndrome, acute myeloid leukemia, or chronic myeloid leukemia.

[0027] In some embodiments, the first antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 36, a light chain CDR2 (CDRL2) of SEQ ID NO: 37, and a light chain CDR3 (CDRL3) of SEQ ID NO: 38, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 33, a heavy chain CDR2 of SEQ ID NO: 34, and a heavy chain CDR3 of SEQ ID NO: 35. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 42, a light chain CDR2 (CDRL2) of SEQ ID NO: 43, and a light chain CDR3 (CDRL3) of SEQ ID NO: 44, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 39, a heavy chain CDR2 of SEQ ID NO: 40, and a heavy chain CDR3 of SEQ ID NO: 41. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 48, a light chain CDR2 (CDRL2) of SEQ ID NO: 49, and a light chain CDR3 (CDRL3) of SEQ ID NO: 50, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 45, a heavy chain CDR2 of SEQ ID NO: 46, and a heavy chain CDR3 of SEQ ID NO: 47.

[0028] In some embodiments, the first antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 28 and a heavy chain variable (VH) region of SEQ ID NO: 27. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 30 and a heavy chain variable (VH) region of SEQ ID NO: 29. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 32 and a heavy chain variable (VH) region of SEQ ID NO: 31.

[0029] In some embodiments, the first antigen and the second antigen are macrophage cell lineage markers, hi some embodiments, the first antigen is CD117, CD34, or CD123, and the second antigen is CD163.

[0030] In some embodiments, the TFC is a metastatic TFC. In some embodiments, the bispecific molecule is a bispecific antibody or antigen-binding fragment thereof.

[0031] In some embodiments, the bispecific antibody is conjugated to a drug. In some embodiments, the drug is a toxin. In some embodiments, the drug is a chemotherapeutic agent.

[0032] In some embodiments, the bispecific molecule comprises a dual nanobody, a BiTE, a tandAb, a DART, a DART-Fc, a DARPin, a scFv, a scFv-HAS-scFV, and a DNL-Fab3. In some embodiments, the bispecific molecule is a bispecific chimeric antigen receptor (CAR). In some embodiments, the bispecific molecule is a Co-LOCKR comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer. In some embodiments, the first polypeptide comprises SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 19. In some embodiments, the second polypeptide comprises SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 21.

[0033] In some embodiments, the bispecific CAR is a synNotch CAR. In some embodiments, the bispecific molecule is a Co-LOCKR comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer, and the third polypeptide binds to the bispecific CAR, and the third polypeptide is operably linked to the first polypeptide or the second polypeptide.

[0034] In some embodiments, the bispecific molecule comprises a split CAR-T system comprising a chimeric antigen receptor (CAR) module and a chimeric costimulatory receptor (CCR) module, wherein the CAR module comprises a polypeptide comprising a first antigen binding region and a CD3z signaling domain, and the CCR module comprises a polypeptide comprising a second antigen binding region and two or more costimulatory domains, and wherein the CAR module and the CCR module each bind to a different antigen on the TFC of the cancer. In some embodiments, the split CAR-T system comprises one or more of the polypeptide sequences in Table 4.

[0035] In certain aspects, the subject matter described herein provides modified cells that express at least one epithelial cell lineage marker and at least one macrophage cell lineage marker.

[0036] In some embodiments, the at least one epithelial cell lineage marker is any one of the markers in Figure 2. In some embodiments, the at least one epithelial cell lineage marker is an epithelial cell adhesion molecule (EpCAM). In some embodiments, EpCAM comprises SEQ ID NO:7 or SEQ ID NO:12.

[0037] In some embodiments, the at least one marker of macrophage cell lineage is any one of the markers in Figure 1. In some embodiments, the at least one marker of macrophage cell lineage is CD163. In some embodiments, CD163 comprises SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:13.

[0038] In certain aspects, the subject matter described herein provides a method of diagnosing cancer, the method comprising detecting cells expressing at least one epithelial cell lineage marker and at least one macrophage cell lineage marker.

[0039] In some embodiments, the at least one epithelial cell lineage marker is any one of the markers in Figure 2. In some embodiments, the at least one epithelial cell lineage marker is epithelial cell adhesion molecule (EpCAM). In some embodiments, EpCAM comprises SEQ ID NO:7.

[0040] In some embodiments, the at least one marker of macrophage cell lineage is any one of the markers in Figure 1. In some embodiments, the at least one marker of macrophage cell lineage is CD163. In some embodiments, CD163 comprises SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0041] In some embodiments, detecting comprises an assay in which the bispecific molecule binds to at least one epithelial cell lineage marker and at least one macrophage cell lineage marker.

[0042] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is breast cancer, brain cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, liver cancer, lung cancer, thymus cancer, ovarian cancer, prostate cancer, or endometrial cancer. In some embodiments, the gastrointestinal cancer is gastric cancer or colon cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the liquid cancer is a leukemia, lymphoma, or myeloma. In some embodiments, the liquid cancer is acute myeloid leukemia (AML). In some embodiments, the liquid cancer is a B-cell malignancy. In some embodiments, the liquid cancer is a myeloid neoplasm, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS / MPN overlap syndrome, acute myeloid leukemia, or chronic myeloid leukemia.

[0043] One or more of the figures are presented in color. To comply with PCT patent application requirements, many of the figures presented herein are black-and-white renditions of images originally produced in color. [Brief description of the drawings]

[0044] [Figure 1-1] Antigens that are predominantly or exclusively macrophage expressed are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Figure 1-2] Antigens that are predominantly or exclusively macrophage expressed are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Figure 1-3] Antigens that are predominantly or exclusively macrophage expressed are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Figure 1-4] Antigens that are predominantly or exclusively macrophage expressed are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Figure 1-5] Antigens that are predominantly or exclusively macrophage expressed are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Figure 1-6] Antigens that are predominantly or exclusively macrophage expressed are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Figure 1-7] Antigens that are predominantly or exclusively macrophage expressed are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Figure 1-8] Antigens that are predominantly or exclusively macrophage expressed are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Figure 1-9]Antigens that are predominantly or exclusively macrophage expressed are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Figure 1-10] Antigens that are predominantly or exclusively macrophage expressed are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Figure 2-1] Antigens that express predominantly or exclusively epithelia are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Figure 2-2] Antigens that express predominantly or exclusively epithelia are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Figure 2-3] Antigens that express predominantly or exclusively epithelia are indicated. In the last two columns, "+" indicates the presence of expression, "-" indicates the absence of expression, and blank indicates unknown expression. [Diagram 3] 1 shows one embodiment of Co-LOCKR. A shows the conformational change of Co-LOCKR. B shows Co-LOCKR recruiting CAR-T cells. [Figure 4] 1 shows separation of cells by size with larger cells being selected for. [Diagram 5] Schematic diagrams of cells expressing no lineage specific antigens (LSA) (A), cells expressing each LSA alone (B and C), or cells expressing both antigens together (D). Cells lacking or expressing individual LSA expression are used as controls to demonstrate the specificity of the different modalities tested against the target. [Figure 6A]Flow cytometry analysis of expression of CD163 (A) or EpCAM (B), or both CD163 and EpCAM (C). The left panel shows isotype control, and the right panel shows staining with antibodies recognizing EpCAM (clone 9C4, PerCP / Cyanine5.5 mouse monoclonal IgG2; Catalog No. 324213 Biolegend) or CD163 (clone RM3 / 1, PE-conjugated mouse monoclonal IgG2; Catalog No. 326506 Biolegend). [Figure 6B] Flow cytometry analysis of expression of CD163 (A) or EpCAM (B), or both CD163 and EpCAM (C). The left panel shows isotype control, and the right panel shows staining with antibodies recognizing EpCAM (clone 9C4, PerCP / Cyanine5.5 mouse monoclonal IgG2; Catalog No. 324213 Biolegend) or CD163 (clone RM3 / 1, PE-conjugated mouse monoclonal IgG2; Catalog No. 326506 Biolegend). [Figure 6C] Flow cytometry analysis of expression of CD163 (A) or EpCAM (B), or both CD163 and EpCAM (C). The left panel shows isotype control, and the right panel shows staining with antibodies recognizing EpCAM (clone 9C4, PerCP / Cyanine5.5 mouse monoclonal IgG2; Catalog No. 324213 Biolegend) or CD163 (clone RM3 / 1, PE-conjugated mouse monoclonal IgG2; Catalog No. 326506 Biolegend). [Figure 7] Schematics of the binding of the cage and key, respectively, to cells expressing either antigen A (panel A) or antigen B (panel B). When the key is not co-localized with the cage, the cage is in a closed conformation and sequesters the latch (panel A), whereas the latch is released from the cage when the key and cage are co-localized with antigens A and B on the same cell (panel C). [Figure 8]Schematic diagrams of the various components of the Co-LOCKR components, Cage and Key proteins (Panel A), or variants of Cage and Key proteins (Panel B). Antigen binding domains for A or B proteins are created in combinations of both Cage and Key to identify optimal combinations. [Figure 9] Expression and purification of Co-LOCKR protein. A) Coomassie G250 stained gel. B) Immunoblot using anti-HIs6 antibody. 1) PageRuler™ Plus prestained protein ladder. 2) His6_TEV_EpCAM-ScFV_Cage (~64kDa). 3) His6_TEV_Key_EpCAM-ScFV (~38kDa). 4) His6_TEV_Key_N3_EpCAM-ScFV (~37kDa). 5) PageRuler™ Plus prestained protein ladder. 6) His6_TEV_CD163-ScFV_Cage (~62kDa). 7) His6_TEV_Key_CD163-ScFV (~35.8kDa). 8) His6_TEV_CD163-ScFV_Cage_I287A (~62kDa). 9.His6_TEV_Key_N3_CD163-ScFV (approximately 35.5 kDa). [Figure 10] 1 shows a schematic diagram of binding of split CAR-T cells expressing CAR and CCR modules. When split CAR-T binds to a target cell expressing both antigens A and B, it is activated and co-stimulated, resulting in eradication of cells expressing A and B. When split CAR-T binds to cells expressing either A or B, suboptimal activation occurs. [Figure 11] 1 shows a schematic diagram of the various components of the CAR and CCR modules of the split CAR-T system. Antigen binding domains to A or B proteins are created in both CAR and CCR combinations to identify optimal combinations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] In certain aspects, the subject matter described herein provides bispecific molecules comprising at least two antigen-binding regions, each antigen-binding region binding to a different antigen on the cancer's first cell (TFC).

[0046] In some embodiments, the first antigen is an epithelial cell lineage marker. In some embodiments, the epithelial cell lineage marker is any one of the markers in FIG. 2. In some embodiments, the epithelial cell lineage marker is epithelial cell adhesion molecule (EpCAM). In some embodiments, EpCAM comprises SEQ ID NO:7 or SEQ ID NO:12. In some embodiments, the second antigen is a macrophage cell lineage marker. In some embodiments, the macrophage cell lineage marker is any one of the markers in FIG. 1. In some embodiments, the macrophage cell lineage marker is CD163. In some embodiments, CD163 comprises SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:13.

[0047] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is breast cancer, brain cancer, gastrointestinal cancer, pancreatic cancer, renal cancer, liver cancer, lung cancer, thymus cancer, ovarian cancer, prostate cancer, or endometrial cancer. In some embodiments, the gastrointestinal cancer is gastric cancer or colon cancer.

[0048] In some embodiments, the cancer comprises a liquid cancer. In some embodiments, the liquid cancer is a leukemia, lymphoma, or myeloma. In some embodiments, the liquid cancer is acute myeloid leukemia (AML). In some embodiments, the liquid cancer is a B-cell malignancy. In some embodiments, the liquid cancer is a myeloid neoplasm, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS / MPN overlap syndrome, acute myeloid leukemia, or chronic myeloid leukemia.

[0049] In some embodiments, the first antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 36, a light chain CDR2 (CDRL2) of SEQ ID NO: 37, and a light chain CDR3 (CDRL3) of SEQ ID NO: 38, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 33, a heavy chain CDR2 of SEQ ID NO: 34, and a heavy chain CDR3 of SEQ ID NO: 35. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 42, a light chain CDR2 (CDRL2) of SEQ ID NO: 43, and a light chain CDR3 (CDRL3) of SEQ ID NO: 44, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 39, a heavy chain CDR2 of SEQ ID NO: 40, and a heavy chain CDR3 of SEQ ID NO: 41. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 48, a light chain CDR2 (CDRL2) of SEQ ID NO: 49, and a light chain CDR3 (CDRL3) of SEQ ID NO: 50, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 45, a heavy chain CDR2 of SEQ ID NO: 46, and a heavy chain CDR3 of SEQ ID NO: 47.

[0050] In some embodiments, the first antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 28 and a heavy chain variable (VH) region of SEQ ID NO: 27. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 30 and a heavy chain variable (VH) region of SEQ ID NO: 29. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 32 and a heavy chain variable (VH) region of SEQ ID NO: 31.

[0051] In some embodiments, the first antigen and the second antigen are macrophage cell lineage markers. In some embodiments, the first antigen is CD117, CD34, or CD123, and the second antigen is CD163. In some embodiments, the TFC is a metastatic TFC.

[0052] In some embodiments, the bispecific molecule is a bispecific antibody or an antigen-binding fragment thereof. In some embodiments, the bispecific antibody is conjugated to a drug. In some embodiments, the drug is a toxin. In some embodiments, the drug is a chemotherapeutic agent. In some embodiments, the bispecific molecule comprises a dual nanobody, a BiTE, a tandAb, a DART, a DART-Fc, a DARPin, a scFv, a scFv-HAS-scFV, and a DNL-Fab3. In some embodiments, the bispecific molecule is a bispecific chimeric antigen receptor (CAR). In some embodiments, the bispecific molecule is a Co-LOCKR comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer.

[0053] In some embodiments, the first polypeptide comprises SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 19. In some embodiments, the second polypeptide comprises SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO:21.

[0054] In some embodiments, the bispecific CAR is a synNotch CAR. In some embodiments, the bispecific molecule is Co-LOCKR comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer, and the third polypeptide binds to the CAR, and the third polypeptide is operably linked to the first polypeptide or the second polypeptide.

[0055] In some embodiments, the bispecific molecule comprises a split CAR-T system comprising a chimeric antigen receptor (CAR) module and a chimeric costimulatory receptor (CCR) module, wherein the CAR module comprises a polypeptide comprising a first antigen binding region and a CD3z signaling domain, and the CCR module comprises a polypeptide comprising a second antigen binding region and two or more costimulatory domains, and wherein the CAR module and the CCR module each bind to a different antigen on the TFC of the cancer. In some embodiments, the split CAR-T system comprises one or more of the polypeptide sequences in Table 4.

[0056] In certain aspects, the subject matter described herein provides a pharmaceutical composition comprising a bispecific molecule according to any embodiment described herein.

[0057] In certain aspects, the subject matter described herein provides a polynucleotide encoding a bispecific molecule according to any embodiment described herein.

[0058] In certain aspects, the subject matter described herein provides a vector comprising a polynucleotide according to any embodiment described herein.

[0059] In certain aspects, the subject matter described herein provides a virus comprising a polynucleotide according to any embodiment described herein.

[0060] In certain aspects, the subject matter described herein provides a genetically modified cell comprising a bispecific molecule according to any embodiment described herein.

[0061] In certain aspects, the subject matter described herein provides a genetically modified cell comprising a polynucleotide according to any embodiment described herein.

[0062] In certain aspects, the subject matter described herein provides a method of treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject a bispecific molecule comprising at least two antigen binding regions, each antigen binding region binding to a different antigen on a cancer cell of origin (TFC).

[0063] In some embodiments, the first antigen is an epithelial cell lineage marker. In some embodiments, the epithelial cell lineage marker is any one of the markers in FIG. 2. In some embodiments, the epithelial cell lineage marker is epithelial cell adhesion molecule (EpCAM). In some embodiments, EpCAM comprises SEQ ID NO:7 or SEQ ID NO:12. In some embodiments, the second antigen is a macrophage cell lineage marker. In some embodiments, the macrophage cell lineage marker is any one of the markers in FIG. 1. In some embodiments, the macrophage cell lineage marker is CD163. In some embodiments, CD163 comprises SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:13.

[0064] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is breast cancer, brain cancer, gastrointestinal cancer, pancreatic cancer, renal cancer, liver cancer, lung cancer, thymic cancer, ovarian cancer, prostate cancer, or endometrial cancer. In some embodiments, the gastrointestinal cancer is gastric cancer or colon cancer.

[0065] In some embodiments, the cancer is a liquid cancer. In some embodiments, the liquid cancer is a leukemia, lymphoma, or myeloma. In some embodiments, the liquid cancer is acute myeloid leukemia (AML). In some embodiments, the liquid cancer is a B-cell malignancy. In some embodiments, the liquid cancer is a myeloid neoplasm, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS / MPN overlap syndrome, acute myeloid leukemia, or chronic myeloid leukemia.

[0066] In some embodiments, the first antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 36, a light chain CDR2 (CDRL2) of SEQ ID NO: 37, and a light chain CDR3 (CDRL3) of SEQ ID NO: 38, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 33, a heavy chain CDR2 of SEQ ID NO: 34, and a heavy chain CDR3 of SEQ ID NO: 35. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 42, a light chain CDR2 (CDRL2) of SEQ ID NO: 43, and a light chain CDR3 (CDRL3) of SEQ ID NO: 44, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 39, a heavy chain CDR2 of SEQ ID NO: 40, and a heavy chain CDR3 of SEQ ID NO: 41. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 48, a light chain CDR2 (CDRL2) of SEQ ID NO: 49, and a light chain CDR3 (CDRL3) of SEQ ID NO: 50, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 45, a heavy chain CDR2 of SEQ ID NO: 46, and a heavy chain CDR3 of SEQ ID NO: 47.

[0067] In some embodiments, the first antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 28 and a heavy chain variable (VH) region of SEQ ID NO: 27. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 30 and a heavy chain variable (VH) region of SEQ ID NO: 29. In some embodiments, the second antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 32 and a heavy chain variable (VH) region of SEQ ID NO: 31.

[0068] In some embodiments, the first antigen and the second antigen are macrophage cell lineage markers, hi some embodiments, the first antigen is CD117, CD34, or CD123, and the second antigen is CD163.

[0069] In some embodiments, the TFC is a metastatic TFC. In some embodiments, the bispecific molecule is a bispecific antibody or antigen-binding fragment thereof.

[0070] In some embodiments, the bispecific antibody is conjugated to a drug. In some embodiments, the drug is a toxin. In some embodiments, the drug is a chemotherapeutic agent.

[0071] In some embodiments, the bispecific molecule comprises a dual nanobody, a BiTE, a tandAb, a DART, a DART-Fc, a DARPin, a scFv, a scFv-HAS-scFV, and a DNL-Fab3. In some embodiments, the bispecific molecule is a bispecific chimeric antigen receptor (CAR). In some embodiments, the bispecific molecule is a Co-LOCKR comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer. In some embodiments, the first polypeptide comprises SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 19. In some embodiments, the second polypeptide comprises SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 21.

[0072] In some embodiments, the bispecific CAR is a synNotch CAR. In some embodiments, the bispecific molecule is a Co-LOCKR comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer, and the third polypeptide binds to the bispecific CAR, and the third polypeptide is operably linked to the first polypeptide or the second polypeptide.

[0073] In some embodiments, the bispecific molecule comprises a split CAR-T system comprising a chimeric antigen receptor (CAR) module and a chimeric costimulatory receptor (CCR) module, wherein the CAR module comprises a polypeptide comprising a first antigen binding region and a CD3z signaling domain, and the CCR module comprises a polypeptide comprising a second antigen binding region and two or more costimulatory domains, and wherein the CAR module and the CCR module each bind to a different antigen on the TFC of the cancer. In some embodiments, the split CAR-T system comprises one or more of the polypeptide sequences in Table 4.

[0074] In certain aspects, the subject matter described herein provides modified cells that express at least one epithelial cell lineage marker and at least one macrophage cell lineage marker.

[0075] In some embodiments, the at least one epithelial cell lineage marker is any one of the markers in Figure 2. In some embodiments, the at least one epithelial cell lineage marker is an epithelial cell adhesion molecule (EpCAM). In some embodiments, EpCAM comprises SEQ ID NO:7 or SEQ ID NO:12.

[0076] In some embodiments, the at least one marker of macrophage cell lineage is any one of the markers in Figure 1. In some embodiments, the at least one marker of macrophage cell lineage is CD163. In some embodiments, CD163 comprises SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:13.

[0077] In certain aspects, the subject matter described herein provides a method of diagnosing cancer, the method comprising detecting cells expressing at least one epithelial cell lineage marker and at least one macrophage cell lineage marker.

[0078] In some embodiments, the at least one epithelial cell lineage marker is any one of the markers in Figure 2. In some embodiments, the at least one epithelial cell lineage marker is epithelial cell adhesion molecule (EpCAM). In some embodiments, EpCAM comprises SEQ ID NO:7.

[0079] In some embodiments, the at least one marker of macrophage cell lineage is any one of the markers in Figure 1. In some embodiments, the at least one marker of macrophage cell lineage is CD163. In some embodiments, CD163 comprises SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0080] In some embodiments, detecting comprises an assay in which the bispecific molecule binds to at least one epithelial cell lineage marker and at least one macrophage cell lineage marker.

[0081] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is breast cancer, brain cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, liver cancer, lung cancer, thymus cancer, ovarian cancer, prostate cancer, or endometrial cancer. In some embodiments, the gastrointestinal cancer is gastric cancer or colon cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the liquid cancer is a leukemia, lymphoma, or myeloma. In some embodiments, the liquid cancer is acute myeloid leukemia (AML). In some embodiments, the liquid cancer is a B-cell malignancy. In some embodiments, the liquid cancer is a myeloid neoplasm, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS / MPN overlap syndrome, acute myeloid leukemia, or chronic myeloid leukemia.

[0082] Specific targeting of cancer cells is a major challenge in the cancer field. 1The most common approaches to cancer treatment, such as chemotherapy and radiation therapy, are indiscriminate, targeting both cancer cells and normal cells (non-cancerous or healthy cells), causing patients to experience many painful side effects. Targeted approaches to kill cancer cells, such as immunotherapy approaches including antibody and chimeric antigen therapy (CAR-T), have the ability to precisely eliminate cells that express one or more pre-determined antigens. However, the biggest challenge is to identify antigens unique to cancer cells that can be targeted. 1、2 This challenge stems from the lack of a single unique antigen that is present only in cancer cells and not in normal cells. Strategies to differentiate cancer cells include targeting two or more antigens. 1 .

[0083] In some embodiments, the subject matter disclosed herein relates to discoveries that do not occur naturally, most of which are not. When an organ or tissue is stressed, the stressed cells can develop heroic survival strategies. In some embodiments, one of these cell survival strategies involves fusion with blood-borne macrophages. 3~6 This hybrid tissue cell and macrophage, called the tumor starter cell (TFC), initiates the cancerous growth. Thus, cancer does not necessarily start with one cell, but can start with two cells.

[0084] In some embodiments, this TFC undergoes genomic rearrangements and re-modifications, resulting in multiple outcomes including:

[0085] In some embodiments, the TFC have the ability to evade the immune system - the TFC express macrophage markers and therefore can evade the immune system. 4、5、7~13 .

[0086] In some embodiments, the TFC is a hybrid, which retains properties of both tissues of origin. Because the TFC is a part of a macrophage, it can move freely throughout the body and may be highly associated with metastasis. 4、5、7~13 .

[0087] In some embodiments, TFCs can be visually identified. In some embodiments, TFCs appear as giant polyploid cells. In some embodiments, TFCs can be observed as giant polyploid cells in 100% of solid tumors. In some embodiments, TFCs can be observed as giant polyploid cells in cases of myelodysplastic syndromes (MDS). In some embodiments, TFCs can be observed as giant polyploid cells in cases of acute myeloid leukemia (AML). 7 .

[0088] In solid tumors, TFCs may express at least one marker of the epithelial tissue from which they originate and at least one marker of macrophages 5、14 In some embodiments, the epithelial tissue marker is epithelial cell adhesion molecule (EpCAM). EpCAM is present on epithelial cells that line the surfaces and cavities of the body. EpCAM spans the membrane of epithelial cells and is important for cell adhesion. In some embodiments, the macrophage marker is CD163, a scavenger receptor for the haptoglobin-hemoglobin complex.

[0089] In some embodiments, the TFC is distinct from other tumor cells. In some embodiments, the TFC is distinct from circulating tumor cells (CTCs). 7 .

[0090] In some embodiments, TFCs are also referred to as CAML (cancer associated macrophage-like cells) or PACC (polyploid cancer cells) or PGCC (polyploid giant cancer cells), etc. 7 In some embodiments, TFCs may be referred to as syncytial or giant cells.

[0091] In some embodiments, the TFC is unique in that it expresses the EpCAM and CD163 markers. 7Expression of EpCAM is restricted to cells of the epithelial lineage, and expression of CD163 is restricted to cells of the macrophage lineage. In some embodiments, no normal cells express both of these antigens.

[0092] In some embodiments, the subject matter described herein relates to targeting two antigens to eliminate TFCs as part of a patient's cancer therapy. In some embodiments, the antigens are an epithelial cell lineage marker and a macrophage cell lineage marker. In some embodiments, the antigens are EpCAM and CD163. In some embodiments, the two antigens are targeted by any of the bispecific molecules that recognize the two antigens described herein, including but not limited to bispecific antibodies that recognize the two antigens.

[0093] In some embodiments, a Boolean AND system can be used to avoid targeting cells that express only one of these antigens. In some embodiments, this system targets only TFCs since both antigens (EpCAM and CD163) are present. In some embodiments, this strategy misses cells that express only either CD163 (macrophage lineage) or EpCAM (epithelial lineage).

[0094] Boolean operators form the basis of mathematical sets and database logic to narrow or widen search results by combining search terms. There are three basic Boolean operators: AND, OR, and NOT. Using the AND operator will narrow search results as all search terms must be present in the resulting records. Logic-based models with only two states are called Boolean models. In some embodiments, the principles of Boolean logic gates, primarily used in the design and function of integrated circuits, are implemented herein to sense one or more inputs and integrate these inputs to generate a desired biological output. In some embodiments, a logical AND gate generates an output if all specified inputs are present. In some embodiments of the subject matter disclosed herein, a logical AND gate is implemented in the design of a molecular circuit. In some embodiments, a cytotoxic response (output) can be enabled only if both antigens (inputs) EpCAM and CD163 are present on the same cell.

[0095] In some embodiments, when targeting two antigens on TFC, one or more of the following approaches can be utilized. In some embodiments, the approach is based on large biomolecules (e.g., proteins). In some embodiments, the approach is based on adoptive cell therapy (e.g., CAR-T).

[0096] In some embodiments, the subject matter disclosed herein relates to a method of selectively targeting cancer cells, the method comprising targeting at least two antigen binding regions, each antigen binding region binding to a different antigen on a cancer cell of origin (TFC). In some embodiments, the first antigen is an epithelial cell lineage marker. In some embodiments, the epithelial cell lineage marker is any one of the markers in FIG. 2. In some embodiments, the epithelial cell lineage marker is epithelial cell adhesion molecule (EpCAM). In some embodiments, the second antigen is a macrophage cell lineage marker. In some embodiments, the macrophage cell lineage marker is any one of the markers in FIG. 1. In some embodiments, the macrophage cell lineage marker is CD163. In some embodiments, the TFC expresses one or more of the markers in FIG. 2 and one or more of the markers in FIG. 1. In some embodiments, for example, in myeloid leukemia or cancer of myeloid origin, the first antigen and the second antigen are both macrophage cell lineage markers. In some embodiments, both markers of macrophage cell lineage are selected from any one of the markers in FIG. 1. In some embodiments, the first antigen is CD117, CD34, CD123, or any combination thereof, and the second antigen is CD163. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is breast cancer, brain cancer, gastrointestinal cancer including gastric cancer and colon cancer, pancreatic cancer, renal cancer, liver cancer, lung cancer, thymic cancer, ovarian cancer, prostate cancer, or endometrial cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the liquid cancer is a leukemia, lymphoma, or myeloma. In some embodiments, the liquid cancer is a B-cell malignancy including, but not limited to, multiple myeloma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma (NHL), or chronic lymphocytic leukemia (CLL). In some embodiments, the liquid cancer is acute myeloid leukemia (AML). In some embodiments, the liquid cancer is a myeloid tumor.In some embodiments, the liquid cancer is myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS / MPN overlap syndrome, acute myeloid leukemia, or chronic myeloid leukemia. In some embodiments, the TFC is a metastatic TFC. In some embodiments, the bispecific molecule is a bispecific antibody or antigen-binding fragment thereof. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is conjugated to a toxin. In some embodiments, the bispecific molecule comprises a dual nanobody, DARPins, BiTE, tandAb, DART, DART-Fc, scFv, scFv-HAS-scFV, and DNL-Fab3. In some embodiments, the bispecific molecule is a bispecific chimeric antigen receptor (CAR). In some embodiments, the bispecific molecule is a co-latching orthogonal cage-key protein (Co-LOCKR) comprising a first polypeptide and a second polypeptide, each of which binds to a different antigen on the TFC of the cancer.

[0097] In some embodiments, the bispecific molecule is Co-LOCKR comprising a first polypeptide, a second polypeptide, and a third polypeptide, where the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer, and the third polypeptide binds to a CAR, where the third polypeptide is operably linked to the first polypeptide or the second polypeptide. In some embodiments, there is a conformational change of the first and second polypeptides after binding to their respective antigens. In some embodiments, the conformational change makes the third polypeptide exposed to surrounding proteins. In some embodiments, the CAR is expressed on the surface of the T cell. In some embodiments, the bispecific CAR is a synNotch CAR. In some embodiments, the bispecific molecule comprises a designed ankyrin repeat protein (DARPin). In some embodiments, the first and second polypeptides of Co-LOCKR use a DARPin domain to bind to their respective antigens (e.g., EpCAM and CD163) on the TFC of the cancer.

[0098] Bispecific molecules of the invention In certain aspects, the present invention provides bispecific molecules comprising at least two antigen-binding regions, each antigen-binding region binding to a different antigen on the cancer's first cell (TFC).

[0099] In some embodiments, the bispecific molecule is a bispecific antibody, a functional equivalent thereof, an antigen-binding fragment thereof, a derivative thereof, or an antibody-like bispecific molecule. Such molecules are known in the art and include, but are not limited to, full-length heavy and light chains, full-length heavy chains, full-length light chains, Fab fragments, single-chain Fv (scFv) fragments, bivalent single-chain antibodies, or diabodies, each of which is a target antigen, a single domain antibody, or one or more peptides specific for a target antigen. Various bispecific molecule formats are known in the art, for example as described in FIG. 2 below: FIG. 2 in Konterman RE et al., Bispecific Antibodies, Drug Discov.Today 20(July(7))(2015)838-847 and FIG. 1 in Suurs FV, ​​et al., A review of bispecific antibodies and antibody constructs in oncology and clinical challenges, Pharmacol.Ther.2019 Sep;201:103-119, the contents of each of which are incorporated herein by reference in their entirety. Bispecific molecules of the invention include, but are not limited to, immunoglobulin (Ig)-like bispecific antibodies and small bispecific molecules, most of which do not have an Fc region (including, but not limited to, binanobodies, DARPins, BiTEs, tandAbs, DARTs, DART-Fc, scFvs, scFv-HAS-scFVs, and DNL-Fab3s). There are also many alternatives, such as affibodies, peptides, and Co-LOCKR. In fact, as those skilled in the art will appreciate, almost any molecule that binds with high affinity to a particular antigen on the TFC of a cancer can be used as an antigen-binding region. Methods for determining whether a bispecific molecule binds with high affinity to a particular antigen are known to those skilled in the art, and include, but are not limited to, direct and indirect solid-phase assays, such as ELISA and Biacore.

[0100] The structural nature of IgG antibodies is that they have two antigen-binding sites, both of which are specific for the same epitope. Therefore, they are monospecific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Bispecific antibodies are widely applied in tumor immunotherapy because their clinical therapeutic effects may be superior to monoclonal antibodies. In some embodiments, the subject matter described herein relates to a bispecific antibody, its functional equivalent, its antigen-binding fragment, its derivative, or an antibody-like bispecific molecule, which binds to two different antigens on cancer cells. In some embodiments, the bispecific antibody, its functional equivalent, its antigen-binding fragment, its derivative, or antibody-like bispecific molecule, which binds to two different antigens on the surface of the TFC. In some embodiments, one antigen is an epithelial cell lineage marker and the other antigen is a macrophage cell lineage marker. In some embodiments, the epithelial cell lineage marker is EpCAM. In some embodiments, the macrophage cell lineage marker is CD163. In some embodiments, the bispecific antibody, functional equivalent thereof, antigen-binding fragment thereof, derivative thereof, or antibody-like bispecific molecule binds to two different antigens, one selected from FIG. 1 and the other selected from FIG. 2. In some embodiments, both antigens are macrophage cell lineage markers. In some embodiments, the first antigen is CD117, CD34, CD123, or any combination thereof, and the second antigen is CD163. In some embodiments, the bispecific antibody, functional equivalent thereof, antigen-binding fragment thereof, derivative thereof, or antibody-like bispecific molecule binds to two different antigens selected from FIG. 1.

[0101] In some embodiments, the present invention provides bispecific molecules comprising two antigen-binding regions, a first antigen-binding region that binds EpCAM and a second antigen-binding region that binds CD163. Antigen-binding regions specific for EpCAM are described herein, including but not limited to, in the section entitled "Antigen-binding regions or domains specific for EpCAM". Antigen-binding regions specific for CD163 are described herein, including but not limited to, in the section entitled "Antigen-binding regions or domains specific for CD163". Bispecific molecules of the present invention comprise any of the antigen-binding regions specific for EpCAM in combination with any of the antigen-binding regions specific for CD163.

[0102] There are various platforms for generating different kinds of bispecific antibodies. Some techniques for generating bispecific antibodies are based on heterologous recombination of heavy and light chains. Various strategies for generating bispecific antibodies derived from the antigen-binding sites of two different antibodies are known in the art, for example as described in: Konterman RE et al., Bispecific Antibodies, Drug Discov.Today 20(July(7))(2015) 838-847, the contents of which are incorporated herein by reference in their entirety.

[0103] In certain aspects, also provided are pharmaceutical compositions comprising the above-described bispecific molecules, polynucleotides encoding the bispecific molecules, vectors comprising a polynucleotide encoding the bispecific molecules, viruses comprising a polynucleotide encoding the bispecific molecules, genetically modified cells, transformed or transduced host cells comprising the bispecific molecules and / or polynucleotides encoding the bispecific molecules.

[0104] CAR-T therapy Chimeric antigen receptor technology (CAR-T) therapy is a type of cancer treatment in which a patient's own T cells are programmed to attack cancer cells in the body. In this therapy, T cells can be harvested from the patient's blood. Blood from a vein in the patient's arm is passed through an apheresis machine, which removes white blood cells, including the T cells, and the remaining blood is returned to the patient. Then, in a laboratory environment, the gene for a specific receptor that binds to a specific protein on the patient's cancer cells can be introduced into the harvested T cells. The receptor is called a chimeric antigen receptor (CAR). Large numbers of these modified CAR-T cells can be grown in a laboratory environment and infused into the patient's bloodstream. CAR-T cell therapy has a 30%-40% success rate of producing sustained remission without additional treatment. In some embodiments, the methods described herein can be administered in combination with CAR-T therapy.

[0105] In certain aspects, the bispecific molecule of the present invention is a bispecific CAR comprising at least two antigen binding regions, each antigen binding region binding to a different antigen on the cancer's first cell (TFC). In some embodiments, the bispecific CAR binds to two different antigens on the surface of the TFC. In some embodiments, one antigen is an epithelial cell lineage marker and the other antigen is a macrophage cell lineage marker. In some embodiments, the epithelial cell lineage marker is EpCAM. In some embodiments, the macrophage cell lineage marker is CD163. In some embodiments, the bispecific CAR binds to two different antigens, one selected from FIG. 1 and the other selected from FIG. 2. In some embodiments, both antigens are macrophage cell lineage markers. In some embodiments, the first antigen is CD117, CD34, CD123, or any combination thereof, and the second antigen is CD163. In some embodiments, both antigens are macrophage cell lineage markers. In some embodiments, the bispecific CAR binds to two different antigens selected from FIG. 1.

[0106] In some embodiments, the present invention provides a bispecific CAR comprising two antigen-binding regions, a first antigen-binding region that binds EpCAM and a second antigen-binding region that binds CD163. Antigen-binding regions specific for EpCAM are described herein, including but not limited to the section entitled "Antigen-binding regions or domains specific for EpCAM". Antigen-binding regions specific for CD163 are described herein, including but not limited to the section entitled "Antigen-binding regions or domains specific for CD163". The bispecific CAR of the present invention comprises any of the antigen-binding regions specific for EpCAM in combination with any of the antigen-binding regions specific for CD163.

[0107] In certain aspects, pharmaceutical compositions comprising the above-mentioned bispecific CARs, polynucleotides encoding the bispecific CARs, vectors comprising a polynucleotide encoding a bispecific CAR, viruses comprising a polynucleotide encoding a bispecific CAR, and genetically modified cells comprising a bispecific CAR and / or a polynucleotide encoding a bispecific CAR are also provided.

[0108] In some embodiments, approaches that avoid targeting cells that express only one antigen can be used. Such approaches are known in the art and include, but are not limited to, Co-LOCKR, SynNotch CAR cells, and combinatorial antigen recognition systems.

[0109] Co-LOCKR cell targeting Co-LOCKR is a colocalization-dependent protein switch that performs logical operations of AND, OR, and NOT 15The system includes engineered nanoscale devices made of synthetic proteins that target therapeutic agents or antibodies only to cells with a unique, predefined combination of cell surface markers. In some embodiments, these protein switches perform AND logic on the cell surface. In some embodiments, Co-LOCKR proteins perform two-input and three-input logical operations in mixed cell populations. In some embodiments, the Latching Orthogonal Cage-Key Protein (LOCKR) switch is composed of a structural "cage" protein that uses a "latch" domain to sequester a functional peptide in an inactive conformation until binding of another "key" protein induces a conformational change that allows binding to an "effector" protein. In some embodiments, the cage, key, and effector bind in a three-way equilibrium, and the sensitivity of the switch can be tuned by adjusting the relative cage-latch and cage-key affinities. Additional embodiments of the Co-LOCKR system are described in: Lajoie, MJ, et al., Designed protein logic to target cells with precise combinations of surface antigens, Science. 2020 Sep 25;369(6511):1637-1643, which are incorporated herein in their entirety. In some embodiments, the synthetic proteins are molecular switches that have no effect when isolated. However, when they bind at the surface of a target cell, they change conformation and activate molecular beacons. These beacons on the cell surface can induce a pre-determined biological activity (e.g., cell elimination) in a specific target cell. In some embodiments, these molecular beacons recruit CAR-T cells that specifically bind to the molecular beacons, as shown in Figures 3A-B. In some embodiments, the molecular beacons comprise the Bim-Bcl2 system. In some embodiments, Bim is encoded in the latch of the LOCKR system as a sequestered peptide. In some embodiments, Bcl2 is used as an effector of the LOCKR system.

[0110] In some embodiments, the Co-LOCKR system comprises a CAR-T cell that binds to Bcl2, which is used as an effector molecule of the system. In one embodiment, the CAR-T system described herein comprises a Bcl2 CAR sequence comprising the following amino acid sequence:

[0111] In some embodiments, the Bcl2 effector of the LOCKR system is a Bcl CAR having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 1. In some embodiments, the Bcl2 effector of the LOCKR system is a Bcl CAR comprising the sequence of SEQ ID NO: 1. In some embodiments, the Bcl2 effector of the LOCKR system is a Bcl CAR consisting of the sequence of SEQ ID NO: 1. In some embodiments, the Bcl CAR is part of a CAR-T system.

[0112] In some embodiments, the bispecific molecule of the present invention is a Co-LOCKR comprising a first polypeptide and a second polypeptide, where the first polypeptide and the second polypeptide bind to different antigens on a first cell (TFC) of a cancer. In some embodiments, the bispecific molecule is a Co-LOCKR comprising a first polypeptide, a second polypeptide, and a third polypeptide, where the first polypeptide and the second polypeptide each bind to different antigens on a TFC of a cancer, and the third polypeptide binds to a CAR, and the third polypeptide is operably linked to the first polypeptide or the second polypeptide. In some embodiments, one antigen is an epithelial cell lineage marker and the other antigen is a macrophage cell lineage marker. In some embodiments, the subject matter described herein relates to the generation of a bispecific Co-LOCKR system that recognizes EpCAM and CD163. In some embodiments, the epithelial cell lineage marker to which the first polypeptide binds is EpCAM. In some embodiments, the macrophage cell lineage marker to which the second polypeptide binds is CD163. In some embodiments, the first polypeptide binds to any antigen selected from Figure 1 and the second polypeptide binds to any antigen selected from Figure 2. In some embodiments, both antigens are macrophage cell lineage markers. In some embodiments, the first antigen is CD117, CD34, CD123, or any combination thereof, and the second antigen is CD163. In some embodiments, the first polypeptide binds to any antigen selected from Figure 1 and the second polypeptide binds to any other antigen selected from Figure 1. In some embodiments, when the first and second polypeptides bind to the TFC of the cancer, the molecular beacon is activated, for example to induce killer T cells to kill the TFC of the cancer.

[0113] In some embodiments, the present invention provides a bispecific Co-LOCKR comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an antigen binding region that binds EpCAM and the second polypeptide comprises an antigen binding region that binds CD163. Antigen binding regions specific for EpCAM are described herein, including but not limited to, in the section entitled "Antigen binding regions or domains specific for EpCAM". Antigen binding regions specific for CD163 are described herein, including but not limited to, in the section entitled "Antigen binding regions or domains specific for CD163". The bispecific Co-LOCKR of the present invention comprises any of the antigen binding regions specific for EpCAM in combination with any of the antigen binding regions specific for CD163.

[0114] In some embodiments, the present invention provides a bispecific Co-LOCKR comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a key or cage domain and an antigen binding region that binds to EpCAM, and the second polypeptide comprises a key or cage domain and an antigen binding region that binds to CD163, and where the first polypeptide comprises a key domain, the second polypeptide comprises a cage domain, or where the first polypeptide comprises a cage domain, the second polypeptide comprises a key domain. The sequence encoding the bispecific Co-LOCKR may further comprise a signal peptide, a purification tag, and / or a protease site. Those skilled in the art will appreciate that the signal peptide and / or purification tag will be cleaved or removed from any polypeptide prior to administering the bispecific Co-LOCKR to a subject in need thereof. Exemplary key and cage domains, as well as signal peptides, purification tags, and protease sites, are described in Example 2.

[0115] In certain aspects, provided herein are pharmaceutical compositions comprising a Co-LOCKR as described herein, a polynucleotide encoding a Co-LOCKR as described herein, a virus comprising a polynucleotide encoding a Co-LOCKR as described herein, and genetically modified cells comprising a Co-LOCKR as described herein and / or a polynucleotide encoding a Co-LOCKR as described herein. Split CAR-T

[0116] The split chimeric antigen receptor T cell (split CAR-T) system comprises two modules: a module containing a CAR and a module containing a chimeric costimulatory receptor (CCR). Binding of both the CAR and the CCR is required to fully activate the T cell. To achieve balanced signaling and maximal T cell cytotoxic activity against a target cell (e.g., a first cell) expressing two different target antigens, the CAR and CCR can be expressed on the same T cell. In some embodiments, the CAR module comprises an antigen-binding domain that specifically recognizes the first antigen and a polypeptide comprising a CD3z signaling domain. In some embodiments, the CAR module comprises: 1) a signal peptide for membrane targeting (derived from GM-CSF or CD8 alpha), 2) an ScFv sequence (derived from either an anti-EpCAM or anti-CD163 antibody), 3) a hinge region (derived from CD8), 4) a transmembrane domain (derived from CD8), and 5) a CD3z signaling domain.

[0117] In some embodiments, the CCR module comprises a polypeptide comprising an antigen-binding domain that specifically recognizes a second antigen and two or more costimulatory domains. In some embodiments, the CCR module comprises: 1) a signal peptide for membrane targeting (derived from GM-CSF), 2) an ScFv sequence (derived from either an anti-EpCAM antibody or an anti-CD163 antibody), 3) a hinge region (derived from CD28), 4) a transmembrane domain (derived from CD28), and 5) a CD28 costimulatory domain, and 6) a 4-1BB costimulatory domain.

[0118] In some embodiments, the present invention provides a bispecific split CAR-T comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an antigen-binding region that binds to EpCAM, and the second polypeptide comprises an antigen-binding region that binds to CD163. Antigen-binding regions specific for EpCAM are described herein, including but not limited to, in the section entitled "Antigen-binding regions or domains specific for EpCAM". Antigen-binding regions specific for CD163 are described herein, including but not limited to, in the section entitled "Antigen-binding regions or domains specific for CD163". The bispecific split CAR-T of the present invention comprises any of the antigen-binding regions specific for EpCAM in combination with any of the antigen-binding regions specific for CD163.

[0119] In some embodiments, the present invention provides a bispecific split CAR-T comprising a first polypeptide and a second polypeptide, the first polypeptide comprising an antigen-binding region that binds to EpCAM, as well as a spacer domain, a transmembrane domain, and a CD3z signaling domain, and the second polypeptide comprising an antigen-binding region that binds to CD163, a spacer domain, a transmembrane domain, a first costimulatory domain, and a second costimulatory domain. In some embodiments, the present invention provides a bispecific split CAR-T comprising a first polypeptide and a second polypeptide, the first polypeptide comprising an antigen-binding region that binds to CD163, as well as a spacer domain, a transmembrane domain, and a CD3z signaling domain, and the second polypeptide comprising an antigen-binding region that binds to EpCAM, a spacer domain, a transmembrane domain, a first costimulatory domain, and a second costimulatory domain. The polypeptide sequence may further comprise a signal peptide. In some embodiments, the spacer domain is a CD8 hinge domain. In some embodiments, the spacer domain is a CD28 hinge domain. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In some embodiments, the first costimulatory domain is a CD28 costimulatory domain. In some embodiments, the second costimulatory domain is a 4-1BB costimulatory domain. Those skilled in the art will appreciate that signal peptides are cleaved or removed from any polypeptide during post-translational processing. Exemplary spacer domains, transmembrane domains, and costimulatory domains, as well as signal peptides, are described in Example 3.

[0120] In some embodiments, the present invention provides a bispecific split CAR-T comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an antigen binding region that binds EpCAM, as well as a CD8 hinge domain, a CD8 transmembrane domain, and a CD3z signaling domain, and the second polypeptide comprises an antigen binding region that binds CA163, a CD28 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain. In some embodiments, the present invention provides a bispecific split CAR-T comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an antigen binding region that binds CD163, as well as a CD8 hinge domain, a CD8 transmembrane domain, and a CD3z signaling domain, and the second polypeptide comprises an antigen binding region that binds EpCAM, a CD28 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

[0121] In certain aspects, pharmaceutical compositions are also provided comprising a split CAR-T as described herein, a polynucleotide encoding a split CAR-T as described herein, a virus comprising a polynucleotide encoding a split CAR-T as described herein, and a genetically modified cell comprising a split CAR-T as described herein and / or a polynucleotide encoding a split CAR-T as described herein.

[0122] SynNotch CAR cells 16、17 The synthetic Notch (synNotch) pathway can drive pre-determined functional responses in mammalian cells. Individual synNotch pathways do not share common signaling intermediates, making the pathways functionally orthogonal. Multiple synNotch receptors can therefore be used within the same cell to achieve the integration of multiple external cues that comprise a Boolean response program. SynNotch-CAR-T cells are a prime and kill molecular circuit. The synNotch receptor primes and activates the CAR-T cell only if all associated antigens are present on the target cell. This allows the CAR-T cell to target only cancer cells and spare normal cells. An additional CAR-based approach is the SUPRA CAR. 19、20 , RevCAR 21、22 , and AvidCAR 23 In some embodiments, the bispecific CAR is SynNotchCAR. In some embodiments, the TFC is targeted with a bispecific molecule using the Co-LOCKR system of protein switches. In some embodiments, the TFC is targeted with a bispecific molecule using the synCAR system, the SUPRA CAR system, the RevCAR system, or the AvidCAR system. In some embodiments, the TFC is targeted with a bispecific antibody using a combinatorial antigen recognition system. In some embodiments, the TFC is targeted with a bispecific antibody conjugated to a toxin.

[0123] Combinatorial antigen recognition system 18 A combinatorial antigen recognition system can facilitate selective tumor eradication by modified T cells, allowing the modified T cells to become tumor specific even in the absence of truly tumor-specific target antigens.

[0124] DARPins Designed ankyrin repeat proteins (DARPins) are genetically engineered antibody mimetic proteins. DARPins bind to target proteins with high specificity and high affinity. They can be derived from ankyrin repeat proteins, a class of binding proteins involved in diverse cellular functions. In some embodiments, DARPins are composed of two or more repeat polypeptide motifs and have a hydrophobic core protected by an N-terminal cap and a C-terminal cap.

[0125] In some embodiments, the bispecific molecule of the invention is a DARPin molecule comprising at least a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide binding to different antigens on a first cell (TFC) of a cancer. In some embodiments, one antigen is an epithelial cell lineage marker and the other antigen is a macrophage cell lineage marker. In some embodiments, the subject matter described herein relates to generating bispecific DARPin molecules that recognize EpCAM and CD163. In some embodiments, the epithelial cell lineage marker to which the first polypeptide binds is EpCAM. In some embodiments, the macrophage cell lineage marker to which the second polypeptide binds is CD163. In some embodiments, the first polypeptide binds to any antigen selected from FIG. 1 and the second polypeptide binds to any antigen selected from FIG. 2. In some embodiments, both antigens are macrophage cell lineage markers. In some embodiments, the first antigen is CD117, CD34, CD123, or any combination thereof, and the second antigen is CD163. In some embodiments, the first polypeptide binds to any antigen selected from FIG. 1, and the second polypeptide binds to any other antigen selected from FIG. 1. In some embodiments, the bispecific DARPin molecule induces killer T cells to kill TFCs of cancer. In some embodiments, the CAR antigen recognition domain comprises a bispecific DARPin molecule. In some embodiments, the antigen binding domain of the Co-LOCKR system comprises a DARPin. In some embodiments, one or more DARPins that specifically bind to EpCAM and CD163 on target cells are identified by screening one or more DAPRin libraries. In some embodiments, one or more DAPRin libraries are commercially available.

[0126] Single-chain variable fragment A single chain variable fragment (scFv) is a fusion protein of the variable regions of one or more immunoglobulin heavy (VH) and light (VL) chains. The two chains can be linked by a short linker peptide of 10 to about 25 amino acids. The linker can be glycine-rich for flexibility and serine or threonine-rich for solubility. The linker can link the N-terminus of the VH to the C-terminus of the VL, or vice versa. The protein retains the specificity of the original immunoglobulin or immunoglobulins. The scFv can be expressed as the antigen-binding domain of any of the bispecific molecules described herein. The scFv can be expressed as the antigen-binding domain of a CAR. In some embodiments, a multivalent scFv can be designed by linking two or more scFv fragments. In some embodiments, the multivalent scFv can be designed into bispecific tandem scFvs known as bispecific T cell engagers (BiTE antibody constructs).

[0127] In some embodiments, the bispecific molecules of the invention comprise at least two scFvs, each of which has an antigen binding site that binds to a different antigen on a first cell (TFC) of a cancer. In some embodiments, one antigen is an epithelial cell lineage marker and the other antigen is a macrophage cell lineage marker. In some embodiments, the subject matter described herein relates to generating bispecific molecules comprising at least two scFvs that recognize EpCAM and CD163. In some embodiments, the epithelial cell lineage marker that the first scFV binds is EpCAM. In some embodiments, the macrophage cell lineage marker that the second scFV binds is CD163. In some embodiments, the first scFV binds to any antigen selected from FIG. 1 and the second scFV binds to any antigen selected from FIG. 2. In some embodiments, both antigens are macrophage cell lineage markers. In some embodiments, the first antigen is CD117, CD34, CD123, or any combination thereof, and the second antigen is CD163. In some embodiments, the first scFV binds any antigen selected from FIG. 1, and the second scFV binds any other antigen selected from FIG. 1. In some embodiments, the bispecific scFv fragment induces killer T cells to kill the TFC of the cancer. In some embodiments, the CAR antigen recognition domain comprises a bispecific scFv fragment. Embodiments and sequences of antibody molecules that specifically bind to EpCAM are disclosed in U.S. Patent Application Publication No. US 7,632,925 B2, the contents of which are incorporated by reference herein in their entirety. Embodiments and sequences of antibody molecules that specifically bind to CD163 are disclosed in U.S. Patent Application Publication Nos. US2017 / 0119790A1, US9,724,426, and US11,034,770, the contents of each of which are incorporated by reference herein in their entirety.

[0128] Drug-conjugated bispecific antibodies In some embodiments, the subject matter described herein relates to the generation of drug-conjugated bispecific antibodies. In some embodiments, the drug is a toxin. In some embodiments, the toxin is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is emtansine. In some embodiments, the drug is methotrexate, thioguanine, 5-fluorouracil, cytosine arabinoside (ARA-C), cisplatin, actinomycin D, anthracycline, or a vinca alkaloid. In some embodiments, the drug is a microtubule disrupting agent. In some embodiments, the microtubule disrupting agent is an auristatin. In some embodiments, the microtubule disrupting agent comprises a maytansinoid. In some embodiments, the drug is a DNA damaging agent. In some embodiments, the DNA damaging agent is a calicheamicin. In some embodiments, the DNA damaging agent is a duocarmycin. In some embodiments, the DNA damaging agent is doxorubicin. In some embodiments, the toxin kills the target cancer cells. In some embodiments, the drug is conjugated to the bispecific antibody via a biotin-streptavidin interaction. In some embodiments, the drug is covalently conjugated to the bispecific antibody. In some embodiments, the drug is conjugated to the antibody via a linker. Further embodiments of antibody-drug conjugates are disclosed in Khongorzul, P., Antibody-Drug Conjugates: A Comprehensive Review, Mol Cancer Res., 2020, 18(1):3-19 (incorporated herein in its entirety).

[0129] In some embodiments, the goal of drug-conjugated antibody therapy is to deliver highly toxic drugs to their target cells using specific carriers. In some embodiments, such therapy is administered intravenously into the bloodstream to avoid degradation by stomach acid and antibody proteolytic enzymes. In some embodiments, upon recognition of the target cell, the drug-conjugated antibody and its antigen are internalized into the cell by receptor-mediated endocytosis. In some embodiments, upon internalization, free cytotoxic drugs are released into the cytoplasm, where the drugs interfere with cellular machinery, induce apoptosis, and / or ultimately induce cell death.

[0130] In certain aspects, the bispecific molecule of the invention is a drug-conjugated bispecific antibody comprising at least two antigen-binding regions, each antigen-binding region binding to a different antigen on the cancer's first cell (TFC). In some embodiments, the bispecific antibody binds to two different antigens on the surface of the TFC. In some embodiments, one antigen is an epithelial cell lineage marker and the other antigen is a macrophage cell lineage marker. In some embodiments, the epithelial cell lineage marker is EpCAM. In some embodiments, the macrophage cell lineage marker is CD163. In some embodiments, the bispecific antibody binds to two different antigens, one selected from FIG. 1 and the other selected from FIG. 2. In some embodiments, both antigens are macrophage cell lineage markers. In some embodiments, the first antigen is CD117, CD34, CD123, or any combination thereof, and the second antigen is CD163. In some embodiments, the bispecific antibody binds to two different antigens selected from FIG. 1.

[0131] In some embodiments, the present invention provides a drug-conjugated bispecific antibody comprising two antigen-binding regions, a first antigen-binding region that binds EpCAM and a second antigen-binding region that binds CD163. Antigen-binding regions specific for EpCAM are described herein, including but not limited to the section entitled "Antigen-binding regions or domains specific for EpCAM". Antigen-binding regions specific for CD163 are described herein, including but not limited to the section entitled "Antigen-binding regions or domains specific for CD163". The drug-conjugated bispecific antibody of the present invention comprises any of the antigen-binding regions specific for EpCAM in combination with any of the antigen-binding regions specific for CD163.

[0132] In certain aspects, pharmaceutical compositions comprising the above-mentioned drug-conjugated bispecific antibodies, polynucleotides encoding the drug-conjugated bispecific antibodies, vectors comprising the polynucleotides encoding the drug-conjugated bispecific antibodies, viruses comprising the polynucleotides encoding the drug-conjugated bispecific antibodies, and genetically modified cells comprising the bispecific antibodies and / or polynucleotides encoding the drug-conjugated bispecific antibodies are also provided.

[0133] Engineered Hybrid Cells In some embodiments, the subject matter described herein relates to the modification of a cell or cell population that expresses at least one epithelial cell lineage marker and at least one macrophage cell lineage marker. In some embodiments, the at least one epithelial cell lineage marker is any one of the markers in FIG. 2. In some embodiments, the at least one epithelial cell lineage marker is epithelial cell adhesion molecule (EpCAM). In some embodiments, the at least one marker of macrophage cell lineage is any one of the markers in FIG. 1. In some embodiments, the at least one marker of macrophage cell lineage is CD163. In some embodiments, the cells express only either EpCAM or CD163, or both EpCAM and CD163 markers. In some embodiments, the subject matter described herein relates to the modification of a cell or cell population that expresses at least two markers macrophage cell lineage. In some embodiments, the at least two markers are either CD117, CD34, CD123, or any combination thereof and CD163. In some embodiments, the subject matter described herein relates to demonstrating the specific targeting of modified cells expressing both antigens and sparing cells expressing only one of the antigens. In some embodiments, the subject matter described herein relates to performing in vitro and in vivo assay studies to demonstrate the specificity and toxicity of the bispecific molecules. In some embodiments, the target cells used in the assay are modified to express at least one epithelial cell lineage marker and at least one macrophage cell lineage marker. In some embodiments, the target cells used in the assay are engineered to express at least two markers of macrophage cell lineage. In some embodiments, the target cells used in the assay comprise a population of modified cells expressing two different antigens, one selected from FIG. 1 and the other selected from FIG. 2. In some embodiments, the target cells used in the assay comprise a population of modified cells expressing two different antigens selected from FIG. 1.In some embodiments, the target cells used in the assay comprise a population of modified cells that express both EpCAM and CD163 markers. In some embodiments, the subject matter described herein relates to the identification of several other antigens that show restricted lineage-specific expression and can potentially be targeted when the antigen combinations of Figures 1 and 2 are used. In some embodiments, the cancer treatment approaches described herein universally target and eliminate TFCs in all cancers.

[0134] Antigen-binding regions or domains Antibodies are heteromultimeric glycoproteins that contain at least two heavy chains and two light chains. Except for IgM, intact antibodies are usually heterotetrameric glycoproteins composed of two identical light chains (L) and two identical heavy chains (H). Usually, each light chain is linked to a heavy chain by a disulfide bond. Each heavy and light chain has intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end followed by a number of constant regions. Each light chain has a variable domain (VL) and a constant region at the other end. The light chains of antibodies of most vertebrate species can be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of the constant region. The variable domains of an antibody confer binding specificity to the antibody, and certain regions exhibit unique variability, called complementarity determining regions (CDRs). The more conserved portions of the variable regions are called framework regions (FRs). The intact heavy and light chain variable domains of an antibody each contain four FRs bound by three CDRs. The CDRs of each chain are held closely together with the CDRs of the other chain by the FR regions and participate in the formation of the antigen-binding site of the antibody. When preparing a non-human antibody against a specific antigen, the variable region can be humanized by grafting the CDRs from the non-human antibody into the FRs present in the human antibody to be modified. In some embodiments, a humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs of a mouse antibody). In other embodiments, a humanized antibody has one or more CDRs (one, two, three, four, five, or six) that are altered relative to the original antibody, which are also referred to as one or more CDRs derived from one or more CDRs from the original antibody.

[0135] For antigen-binding regions or domains that bind to a desired target derived from antibodies that bind to a desired target (such as, but not limited to, EpCAM and CD163), exemplary amino acid sequences of the variable light (VL) and variable heavy (VH) chains of these antibodies are provided below. Thus, one skilled in the art can construct bispecific molecules having the CDRs of the VH and VL chains, as well as antibodies and derivatives thereof (including humanized derivatives) capable of binding to the epitopes recognized by these antibodies.

[0136] Affinity refers to the equilibrium constant for the reversible binding of two agents and is expressed as KD. In some embodiments, the bispecific molecules and / or antigen-binding regions or domains thereof have affinity in the nanomolar range (10 -7 ~10 -9 In certain embodiments, the antibodies described herein specifically bind to a human CD163 polypeptide with a KD of 10 nM or less. In certain embodiments, the antibodies described herein specifically bind to a human EpCAM polypeptide with a KD of 10 nM or less.

[0137] Antigen-binding region or domain specific for EpCAM The amino acid sequence corresponding to EpCAM (Uniprot ID: P16422) is presented below: MAPPQVLAFGLLLAAATATFAAAQEECVCENYKLAVNCFVNNNRQCQCTSVGAQNTVICSKLAAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSMCWCVNTAGVRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDSKSLRTALQKEITTRYQLDPKFITSILYENNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLTVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKAGVIAVIVVVVIAVVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA (SEQ ID NO: 2).

[0138] In some embodiments, one of the antigen-binding regions of the bispecific molecule binds to EpCAM. In some embodiments, one of the antigen-binding regions of the bispecific molecule binds to the epitope of SEQ ID NO:2. In some embodiments, the invention relates to polynucleotide and polypeptide sequences encoding IgG-based structures, such as heavy and light chain sequences of EpCAM-specific antibodies. Embodiments and sequences of antibody molecules that specifically bind to EpCAM are known in the art, including those disclosed in U.S. Patent No. 7,632,925 B2, the contents of which are incorporated herein by reference in their entirety.

[0139] In some embodiments, the antigen-binding region specific for EpCAM comprises SEQ ID NO:3, which is an EpCAM-specific DARPin: DLGKKLLEAARAGQDDEVRILVANGADVNAYFGTTPLHLAAAHGRLEIVEVLLKNGADVNAQDVWGITPLHLAAYNGHLEIVEVLLKYGADVNAHDTRGWTPLHLAAINGHLEIVEVLLKNVADVNAQDRSGKTPFDLAIDNGNEDIAEVLQKAAKLN (SEQ ID NO: 3).

[0140] In some embodiments, the antigen-binding region specific for EpCAM comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3. Further details of DARPins for EpCAM can be found in: Stefan, N. et al., DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency. J Mol Biol, 2011, 413(4):826-843 (incorporated herein in its entirety).

[0141] In some embodiments, the antigen-binding region specific for EpCAM comprises SEQ ID NO:4, which is a co-LOCKR cage targeted to EpCAM by a DARPin: MGSHHHHHHGSGSENLYFQGSGGSDLGKKLLEAARAGQDDEVRILVANGADVNAYFGTTPLHLAAAHGRLEIVEVLLKNGADVNAQDVWGITPLHLAAYNGHLEIVEVLLKYGADVNAHDTR GWTPLHLAAINGHLEIVEVLLKNVADVNAQDRSGKTPFDLAIDNGNEDIAEVLQKAAKLNSGSGSGKPGQASGSELARKLLEASTKLQRLNIRLAEALLEAIARLQELNLELVYLAVELTDPK RIRDEIKEVKDKSKEIIRRAEKEIDDAAKESEKILEEAREAISGSGSSELAKLLLKAIAETQDLNLRAAKAFLEAAAKLQELNIRAVELLVKLTDPATIREALEHAKRRSKEIIDEAERAIRAAKRESERIIEEARRLIEKGSGSGSELARELLRAHAQLQRLNLELLRELLRALAQLQELNLDLLRLASELTDEIWIAQELRRIGDEFNAYYADAERLIREAAAASEKISREAERLIR (SEQ ID NO: 4).

[0142] In some embodiments, the antigen-binding region specific for EpCAM comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4.

[0143] In some embodiments, the antigen-binding region specific for EpCAM comprises SEQ ID NO:5, which is the key for co-LOCKR targeted to EpCAM by DARPins: MGSHHHHHHGSGSENLYFQGSGGSDEARKAIARVKRESKRIVEDAERLIREAAAASEKISREAERLIRGGGSGSGSGSGKPGQASGSDLGKKLLEAARAGQDDEVRILVANGADVNAYFGTTPLHLAAAHGRLEIVEVLLKNGADVNAQDVWGITPLHLAAYNGHLEIVEVLLKYGADVNAHDTRGWTPLHLAAINGHLEIVEVLLKNVADVNAQDRSGKTPFDLAIDNGNEDIAEVLQKAAKLN (SEQ ID NO: 5).

[0144] In some embodiments, the antigen-binding region specific for EpCAM comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:5.

[0145] In some embodiments, the antigen-binding region specific for EpCAM comprises the VH chain of SEQ ID NO:27: [ka] The CDRs are shown in bold and underlined and include amino acids 31-35 (CDRH1), 50-66 (CDRH2), and 98-117 (CDRH3). A nucleic acid encoding SEQ ID NO:27 is SEQ ID NO:143 of U.S. Patent No. 7,632,925, which is incorporated by reference herein. CDRH1 includes nt 91 to nt 105, CDRH2 includes nt 148 to nt 198, and CDRH3 includes nt 292 to nt 351.

[0146] In some embodiments, the antigen-binding region specific for EpCAM comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:27.

[0147] In some embodiments, the antigen-binding region specific for EpCAM comprises a VH region comprising one or more CDR sequences selected from the following: SYGMH (SEQ ID NO:33) VISYDGSNKYYADSVKG (SEQ ID NO: 34) KDMGWGSGWRPYYYYGMDVW (sequence number 35).

[0148] In some embodiments, the antigen-binding region specific for EpCAM comprises the VL chain of SEQ ID NO:28: [ka] The CDRs are shown in bold and underlined and include amino acids 24-34 (CDR1), 50-56 (CDR2), and 89-98 (CDR3). A nucleic acid encoding SEQ ID NO:28 is SEQ ID NO:147 of U.S. Patent No. 7,632,925, which is incorporated herein by reference. CDRL1 includes nt 70 to nt 102, CDRL2 includes nt 148 to nt 168, and CDRL3 includes nt 265 to nt 294.

[0149] In some embodiments, the antigen-binding region specific for EpCAM comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:28.

[0150] In some embodiments, the antigen-binding region specific for EpCAM comprises a VL region comprising one or more CDR sequences selected from the following: RASQSISSYLN (SEQ ID NO: 36) WASTRES (SEQ ID NO:37) QQSDSLPITF (sequence number 38).

[0151] In some embodiments, the polypeptides described herein are polypeptides that include one or more of the VL and / or VH regions of an antibody that binds to a desired target (such as, but not limited to, EpCAM). In some embodiments, the polypeptides include one or more of the VL and / or VH chain CDRs of an antibody that binds to a desired target (such as, but not limited to, EpCAM). In some embodiments, the polypeptides include three CDRs of the VL and / or VH chains of the antibody. In some embodiments, the polypeptides include an amino acid sequence of an antibody having any of the following: at least 5 contiguous amino acids of the sequence of an antibody that binds to EpCAM, at least 8 contiguous amino acids of an antibody that binds to EpCAM, at least about 10 contiguous amino acids of an antibody that binds to EpCAM, at least about 15 contiguous amino acids of an antibody that binds to EpCAM, at least about 20 contiguous amino acids of an antibody that binds to EpCAM, at least about 25 contiguous amino acids of an antibody that binds to EpCAM, at least about 30 contiguous amino acids of an antibody that binds to EpCAM. In another embodiment, the 5 (or more) contiguous amino acids are from the CDRs of the antibody.

[0152] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence (SEQ ID NO:27).

[0153] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having at least 90% identity to the amino acid sequence (SEQ ID NO:27).

[0154] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having at least 95% identity to the amino acid sequence (SEQ ID NO:27).

[0155] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having at least 99% identity to an amino acid sequence (SEQ ID NO:27).

[0156] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having 100% identity to the amino acid sequence (SEQ ID NO:27). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having at least 80% identity to the amino acid sequence (SEQ ID NO:28). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having at least 90% identity to the amino acid sequence (SEQ ID NO:28). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having at least 95% identity to the amino acid sequence (SEQ ID NO:28). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having at least 99% identity to the amino acid sequence (SEQ ID NO:28). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having 100% identity to the amino acid sequence (SEQ ID NO:28).

[0157] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having at least 80% identity to an amino acid sequence (SEQ ID NO:28).

[0158] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having at least 90% identity to an amino acid sequence (SEQ ID NO:28).

[0159] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having at least 95% identity to an amino acid sequence (SEQ ID NO:28).

[0160] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having at least 99% identity to an amino acid sequence (SEQ ID NO:28).

[0161] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having 100% identity to the amino acid sequence (SEQ ID NO:28). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence (SEQ ID NO:27). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having at least 90% identity to the amino acid sequence (SEQ ID NO:27). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having at least 95% identity to the amino acid sequence (SEQ ID NO:27). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having at least 99% identity to the amino acid sequence (SEQ ID NO:27). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having 100% identity to the amino acid sequence (SEQ ID NO:27).

[0162] In some embodiments, described herein are bispecific molecules comprising a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence (SEQ ID NO:27) and a light chain variable region (VL) having at least 80% identity to the amino acid sequence (SEQ ID NO:28). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having at least 85% identity to the amino acid sequence (SEQ ID NO:28). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having at least 90% identity to the amino acid sequence (SEQ ID NO:28). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having at least 95% identity to the amino acid sequence (SEQ ID NO:28). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having at least 99% identity to the amino acid sequence (SEQ ID NO:28). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having 100% identity to the amino acid sequence (SEQ ID NO:28). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 85% identity to the amino acid sequence (SEQ ID NO:27). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 90% identity to the amino acid sequence (SEQ ID NO:27). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 95% identity to the amino acid sequence (SEQ ID NO:27). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 99% identity to the amino acid sequence (SEQ ID NO:27). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 100% identity to the amino acid sequence (SEQ ID NO:27).

[0163] In some embodiments, described herein are bispecific molecules comprising a heavy chain sequence comprising a complementarity determining region (CDR) H1 having at least 80% identity to the amino acid sequence (SEQ ID NO:33), a CDRH2 having at least 80% identity to the amino acid sequence (SEQ ID NO:34), and a CDRH3 having at least 80% identity to the amino acid sequence (SEQ ID NO:35), and a light chain sequence comprising a CDRL1 having at least 80% identity to the amino acid sequence (SEQ ID NO:36), a CDRL2 having at least 80% identity to the amino acid sequence (SEQ ID NO:37), and a CDRL3 having at least 80% identity to the amino acid sequence (SEQ ID NO:38). In some embodiments, the bispecific molecules comprise a light chain sequence comprising a CDRL1 having at least 85% identity to the amino acid sequence (SEQ ID NO:36), a CDRL2 having at least 85% identity to the amino acid sequence (SEQ ID NO:37), and a CDRL3 having at least 85% identity to the amino acid sequence (SEQ ID NO:38). In some embodiments, the bispecific molecule comprises a light chain sequence comprising a CDRL1 having at least 90% identity to the amino acid sequence (SEQ ID NO:36), a CDRL2 having at least 90% identity to the amino acid sequence (SEQ ID NO:37), and a CDRL3 having at least 90% identity to the amino acid sequence (SEQ ID NO:38). In some embodiments, the bispecific molecule comprises a light chain sequence comprising a CDRL1 having at least 95% identity to the amino acid sequence (SEQ ID NO:36), a CDRL2 having at least 95% identity to the amino acid sequence (SEQ ID NO:37), and a CDRL3 having at least 95% identity to the amino acid sequence (SEQ ID NO:38). In some embodiments, the bispecific molecule comprises a light chain sequence comprising a CDRL1 having at least 99% identity to the amino acid sequence (SEQ ID NO:36), a CDRL2 having at least 99% identity to the amino acid sequence (SEQ ID NO:37), and a CDRL3 having at least 99% identity to the amino acid sequence (SEQ ID NO:38). In some embodiments, the bispecific molecule comprises a light chain sequence comprising a CDRL1 having 100% identity to the amino acid sequence (SEQ ID NO:36), a CDRL2 having 100% identity to the amino acid sequence (SEQ ID NO:37), and a CDRL3 having at least 100% identity to the amino acid sequence (SEQ ID NO:38).In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 85% identity to the amino acid sequence (SEQ ID NO:33), a CDRH2 having at least 85% identity to the amino acid sequence (SEQ ID NO:34), and a CDRH3 having at least 85% identity to the amino acid sequence (SEQ ID NO:35). In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 90% identity to the amino acid sequence (SEQ ID NO:33), a CDRH2 having at least 90% identity to the amino acid sequence (SEQ ID NO:34), and a CDRH3 having at least 90% identity to the amino acid sequence (SEQ ID NO:35). In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 95% identity to the amino acid sequence (SEQ ID NO:33), a CDRH2 having at least 95% identity to the amino acid sequence (SEQ ID NO:34), and a CDRH3 having at least 95% identity to the amino acid sequence (SEQ ID NO:35). In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 99% identity to the amino acid sequence (SEQ ID NO: 33), a CDRH2 having at least 99% identity to the amino acid sequence (SEQ ID NO: 34), and a CDRH3 having at least 99% identity to the amino acid sequence (SEQ ID NO: 35). In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 100% identity to the amino acid sequence (SEQ ID NO: 33), a CDRH2 having at least 100% identity to the amino acid sequence (SEQ ID NO: 34), and a CDRH3 having at least 100% identity to the amino acid sequence (SEQ ID NO: 35).

[0164] In some embodiments, a light chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:36; Described herein are bispecific molecules that bind to EpCAM, comprising at least one of: a light chain CDR2 having an amino acid sequence that is 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; and a light chain CDR3 having an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:38. In some embodiments, a bispecific molecule that binds EpCAM comprises at least one of: a light chain CDR1 having 100% identity to the amino acid sequence set forth as SEQ ID NO:36; a light chain CDR2 having 100% identity to the amino acid sequence set forth as SEQ ID NO:37; and a light chain CDR3 having 100% identity to the amino acid sequence set forth as SEQ ID NO:38.

[0165] In some embodiments, a heavy chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:33; Described herein are bispecific molecules that bind to EpCAM, comprising at least one of: a heavy chain CDR2 having an amino acid sequence that is 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; and a heavy chain CDR3 having an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:35. In some embodiments, a bispecific molecule that binds EpCAM comprises at least one of: a heavy chain CDR1 having 100% identical to the amino acid sequence set forth as SEQ ID NO:33; a heavy chain CDR2 having 100% identical to the amino acid sequence set forth as SEQ ID NO:34; and a heavy chain CDR3 having 100% identical to the amino acid sequence set forth as SEQ ID NO:35.

[0166] In some embodiments, a light chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:36; a light chain CDR2 having an amino acid sequence that is 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:38; a light chain CDR3 having an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:33; a heavy chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:34; , 97%, 98% or 99% identical to the amino acid sequence set forth as SEQ ID NO:35; and a heavy chain CDR3 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth as SEQ ID NO:35.In some embodiments, a bispecific molecule that binds EpCAM comprises at least one of: a light chain CDR1 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 36; a light chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 37; a light chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 38; a heavy chain CDR1 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 33; a heavy chain CDR2 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 34; and a heavy chain CDR3 having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO: 35.

[0167] In some embodiments, bispecific molecules are described herein that contain a VL chain and a VH chain that confer binding specificity to EpCAM, in which amino acids within the framework can be altered, in some embodiments, the amino acid changes include the introduction of conservative amino acid substitutions. In some embodiments, a bispecific molecule that binds EpCAM comprises a light chain variable (VL) region and a heavy chain variable (VH) region; the VL region comprises a light chain CDR1 (CDRL1) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:36, a light chain CDR2 (CDRL2) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:37, and a light chain CDR3 (CDRL3) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:38; and the amino acid sequences of the VL regions outside of CDRL1, CDRL2, and CDRL3 are at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to SEQ ID NO:28. , 98%, or 99% identity; the VH region comprises a heavy chain CDR1 (CDRH1) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:33, a heavy chain CDR2 (CDRH2) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:34, and a heavy chain CDR3 (CDRH3) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:35; and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:27. In some embodiments, the amino acid sequence of the VL region outside of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 90% identity to SEQ ID NO:28, and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 90% identity to SEQ ID NO:27.In some embodiments, the amino acid sequence of the outer VL region of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 95% identity to SEQ ID NO: 28, and the amino acid sequence of the outer VH region of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 95% identity to SEQ ID NO: 27. In some embodiments, the amino acid sequence of the outer VL region of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 99% identity to SEQ ID NO: 28, and the amino acid sequence of the outer VH region of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 99% identity to SEQ ID NO: 27.

[0168] In some embodiments, a bispecific molecule that binds EpCAM comprises a light chain variable (VL) region and a heavy chain variable (VH) region; the VL region comprises a light chain CDR1 (CDRL1) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:36, a light chain CDR2 (CDRL2) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:37, and a light chain CDR3 (CDRL3) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:38; and the amino acid sequences of the VL regions outside of CDRL1, CDRL2, and CDRL3 have an overall sequence identity of at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:28. the V gene usage of the VL region is a kappa light chain; the VH region comprises a heavy chain CDR1 (CDRH1) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:33, a heavy chain CDR2 (CDRH2) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:34, and a heavy chain CDR3 (CDRH3) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:35; the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:27; and the V gene usage of the VH region is an IGHV gene. In some embodiments, the amino acid sequence of the VL region outside of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 90% identity to SEQ ID NO:28, and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 90% identity to SEQ ID NO:27.In some embodiments, the amino acid sequence of the VL region outside of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 95% identity to SEQ ID NO: 28, and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 95% identity to SEQ ID NO: 27. In some embodiments, the amino acid sequence of the VL region outside of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 99% identity to SEQ ID NO: 28, and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 99% identity to SEQ ID NO: 27. In some embodiments, the V gene sequence of the VL region is a kappa 5.1 light chain.

[0169] Antigen-binding regions or domains specific for CD163 The amino acid sequence corresponding to the CD163 (Uniport ID: Q86VB7) sequence is presented below:

[0170] In some embodiments, one of the antigen binding regions of the bispecific molecule binds to CD163. In some embodiments, one of the antigen binding regions of the bispecific molecule binds to the epitope of SEQ ID NO: 6. In some embodiments, the invention relates to polynucleotide and polypeptide sequences encoding IgG-based structures, such as heavy and light chain sequences of CD163-specific antibodies. Embodiments and sequences of antibody molecules that specifically bind to CD163 are known in the art, including those disclosed in U.S. Patent Application Publication No. US2017 / 0119790A1, the contents of which are incorporated herein by reference in their entirety.

[0171] In some embodiments, the polypeptides described herein are polypeptides comprising one or more of the VL and / or VH chain regions of an antibody that binds to a desired target (such as, but not limited to, CD163). In some embodiments, the polypeptide comprises one or more of the VL and / or VH chain CDRs of an antibody that binds to a desired target (such as, but not limited to, CD163). In some embodiments, the polypeptide comprises three CDRs of the VL and / or VH chains of the antibody. In some embodiments, the polypeptide comprises an amino acid sequence of an antibody having any of the following: at least 5 contiguous amino acids of the sequence of an antibody that binds to CD163, at least 8 contiguous amino acids of an antibody that binds to CD163, at least about 10 contiguous amino acids of an antibody that binds to CD163, at least about 15 contiguous amino acids of an antibody that binds to CD163, at least about 20 contiguous amino acids of an antibody that binds to CD163, at least about 25 contiguous amino acids of an antibody that binds to CD163, at least about 30 contiguous amino acids of an antibody that binds to CD163. In another embodiment, the 5 (or more) contiguous amino acids are from the CDRs of the antibody.

[0172] In some embodiments, the antigen-binding region specific for CD163 comprises the VH chain of SEQ ID NO:29: [ka] The CDRs are shown in bold and underlined and include amino acids 26-33 (CDR1), 54-56 (CDR2), and 96-107 (CDR3).

[0173] In some embodiments, the antigen-binding region specific for CD163 comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:29.

[0174] In some embodiments, the antigen binding region specific for CD163 comprises a VH region comprising one or more CDR sequences selected from the following: GYSITSDY (SEQ ID NO: 39) YSG (SEQ ID NO: 40) CVSGTYYFDYWG (sequence number 41).

[0175] In some embodiments, the antigen-binding region specific for CD163 comprises a VL chain of SEQ ID NO: 30: [ka] The CDRs are shown in bold and underlined and include amino acids 25-33 (CDR1), 50-52 (CDR2), and 90-97 (CDR3).

[0176] In some embodiments, the antigen-binding region specific for CD163 comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:30.

[0177] In some embodiments, the antigen binding region specific for CD163 comprises a VL region comprising one or more CDR sequences selected from the following: ASQSVSSDV (SEQ ID NO:42) YAS (SEQ ID NO: 43) QDYTSPRT (sequence number 44).

[0178] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having at least 80% identity to an amino acid sequence (SEQ ID NO:29).

[0179] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having at least 90% identity to an amino acid sequence (SEQ ID NO:29).

[0180] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having at least 95% identity to an amino acid sequence (SEQ ID NO:29).

[0181] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having at least 99% identity to an amino acid sequence (SEQ ID NO:29).

[0182] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having 100% identity to the amino acid sequence (SEQ ID NO:29). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having at least 80% identity to the amino acid sequence (SEQ ID NO:30). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having at least 90% identity to the amino acid sequence (SEQ ID NO:30). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having at least 95% identity to the amino acid sequence (SEQ ID NO:30). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having at least 99% identity to the amino acid sequence (SEQ ID NO:30). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having 100% identity to the amino acid sequence (SEQ ID NO:30).

[0183] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having at least 80% identity to an amino acid sequence (SEQ ID NO:30).

[0184] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having at least 90% identity to an amino acid sequence (SEQ ID NO:30).

[0185] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having at least 95% identity to an amino acid sequence (SEQ ID NO:30).

[0186] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having at least 99% identity to an amino acid sequence (SEQ ID NO:30).

[0187] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having 100% identity to the amino acid sequence (SEQ ID NO:30). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence (SEQ ID NO:29). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having at least 90% identity to the amino acid sequence (SEQ ID NO:29). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having at least 95% identity to the amino acid sequence (SEQ ID NO:29). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having at least 99% identity to the amino acid sequence (SEQ ID NO:29). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having 100% identity to the amino acid sequence (SEQ ID NO:29).

[0188] In some embodiments, described herein are bispecific molecules comprising a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence (SEQ ID NO:29) and a light chain variable region (VL) having at least 80% identity to the amino acid sequence (SEQ ID NO:30). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having at least 85% identity to the amino acid sequence (SEQ ID NO:30). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having at least 90% identity to the amino acid sequence (SEQ ID NO:30). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having at least 95% identity to the amino acid sequence (SEQ ID NO:30). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having at least 99% identity to the amino acid sequence (SEQ ID NO:30). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having 100% identity to the amino acid sequence (SEQ ID NO:30). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 85% identity to the amino acid sequence (SEQ ID NO:29). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 90% identity to the amino acid sequence (SEQ ID NO:29). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 95% identity to the amino acid sequence (SEQ ID NO:29). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 99% identity to the amino acid sequence (SEQ ID NO:29). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 100% identity to the amino acid sequence (SEQ ID NO:29).

[0189] In some embodiments, described herein are bispecific molecules comprising a heavy chain sequence comprising a complementarity determining region (CDR) H1 having at least 80% identity to the amino acid sequence (SEQ ID NO:39), a CDRH2 having at least 80% identity to the amino acid sequence (SEQ ID NO:40), and a CDRH3 having at least 80% identity to the amino acid sequence (SEQ ID NO:41), and a light chain sequence comprising a CDRL1 having at least 80% identity to the amino acid sequence (SEQ ID NO:42), a CDRL2 having at least 80% identity to the amino acid sequence (SEQ ID NO:43), and a CDRL3 having at least 80% identity to the amino acid sequence (SEQ ID NO:44). In some embodiments, the bispecific molecules comprise a light chain sequence comprising a CDRL1 having at least 85% identity to the amino acid sequence (SEQ ID NO:42), a CDRL2 having at least 85% identity to the amino acid sequence (SEQ ID NO:43), and a CDRL3 having at least 85% identity to the amino acid sequence (SEQ ID NO:44). In some embodiments, the bispecific molecule comprises a light chain sequence comprising a CDRL1 having at least 90% identity to the amino acid sequence (SEQ ID NO:42), a CDRL2 having at least 90% identity to the amino acid sequence (SEQ ID NO:43), and a CDRL3 having at least 90% identity to the amino acid sequence (SEQ ID NO:44). In some embodiments, the bispecific molecule comprises a light chain sequence comprising a CDRL1 having at least 95% identity to the amino acid sequence (SEQ ID NO:42), a CDRL2 having at least 95% identity to the amino acid sequence (SEQ ID NO:43), and a CDRL3 having at least 95% identity to the amino acid sequence (SEQ ID NO:44). In some embodiments, the bispecific molecule comprises a light chain sequence comprising a CDRL1 having at least 99% identity to the amino acid sequence (SEQ ID NO:42), a CDRL2 having at least 99% identity to the amino acid sequence (SEQ ID NO:43), and a CDRL3 having at least 99% identity to the amino acid sequence (SEQ ID NO:44). In some embodiments, the bispecific molecule comprises a light chain sequence comprising a CDRL1 having 100% identity to the amino acid sequence (SEQ ID NO: 42), a CDRL2 having 100% identity to the amino acid sequence (SEQ ID NO: 43), and a CDRL3 having at least 100% identity to the amino acid sequence (SEQ ID NO: 44).In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 85% identity to the amino acid sequence (SEQ ID NO:39), a CDRH2 having at least 85% identity to the amino acid sequence (SEQ ID NO:40), and a CDRH3 having at least 85% identity to the amino acid sequence (SEQ ID NO:41). In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 90% identity to the amino acid sequence (SEQ ID NO:39), a CDRH2 having at least 90% identity to the amino acid sequence (SEQ ID NO:40), and a CDRH3 having at least 90% identity to the amino acid sequence (SEQ ID NO:41). In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 95% identity to the amino acid sequence (SEQ ID NO:39), a CDRH2 having at least 95% identity to the amino acid sequence (SEQ ID NO:40), and a CDRH3 having at least 95% identity to the amino acid sequence (SEQ ID NO:41). In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 99% identity to the amino acid sequence (SEQ ID NO: 39), a CDRH2 having at least 99% identity to the amino acid sequence (SEQ ID NO: 40), and a CDRH3 having at least 99% identity to the amino acid sequence (SEQ ID NO: 41). In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 100% identity to the amino acid sequence (SEQ ID NO: 39), a CDRH2 having at least 100% identity to the amino acid sequence (SEQ ID NO: 40), and a CDRH3 having at least 100% identity to the amino acid sequence (SEQ ID NO: 41).

[0190] In some embodiments, a light chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:42; Described herein are bispecific molecules that bind to CD163, comprising at least one of: a light chain CDR2 having an amino acid sequence that is 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; and a light chain CDR3 having an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:44. In some embodiments, bispecific molecules that bind CD163 comprise at least one of: a light chain CDR1 having 100% identity to the amino acid sequence set forth as SEQ ID NO: 42; a light chain CDR2 having 100% identity to the amino acid sequence set forth as SEQ ID NO: 43; and a light chain CDR3 having 100% identity to the amino acid sequence set forth as SEQ ID NO: 44.

[0191] In some embodiments, a heavy chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:39; Described herein are bispecific molecules that bind to CD163, comprising at least one of: a heavy chain CDR2 having an amino acid sequence that is 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; and a heavy chain CDR3 having an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:41. In some embodiments, a bispecific molecule that binds to CD163 comprises at least one of: a heavy chain CDR1 having 100% identity to the amino acid sequence set forth as SEQ ID NO:39; a heavy chain CDR2 having 100% identity to the amino acid sequence set forth as SEQ ID NO:40; and a heavy chain CDR3 having 100% identity to the amino acid sequence set forth as SEQ ID NO:41.

[0192] In some embodiments, a light chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:42; a light chain CDR2 having an amino acid sequence that is 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:44; a light chain CDR3 having an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:39; a heavy chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 40; , 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:41; and a heavy chain CDR3 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth as SEQ ID NO:41.In some embodiments, a bispecific molecule that binds to CD163 comprises at least one of: a light chain CDR1 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 42; a light chain CDR2 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 43; a light chain CDR3 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 44; a heavy chain CDR1 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 39; a heavy chain CDR2 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 40; and a heavy chain CDR3 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 41.

[0193] In some embodiments, bispecific molecules are described herein that comprise a VL chain and a VH chain that confer binding specificity to CD163, in which amino acids within the framework can be altered, hi some embodiments, the amino acid changes comprise the introduction of conservative amino acid substitutions. In some embodiments, a bispecific molecule that binds CD163 comprises a light chain variable (VL) region and a heavy chain variable (VH) region; the VL region comprises a light chain CDR1 (CDRL1) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:42, a light chain CDR2 (CDRL2) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:43, and a light chain CDR3 (CDRL3) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:44; and the amino acid sequences of the VL regions outside of CDRL1, CDRL2, and CDRL3 are at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to SEQ ID NO:30. , 98%, or 99% identity; the VH region comprises a heavy chain CDR1 (CDRH1) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:39, a heavy chain CDR2 (CDRH2) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:40, and a heavy chain CDR3 (CDRH3) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:41; and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:29. In some embodiments, the amino acid sequence of the VL region outside of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 90% identity to SEQ ID NO:30, and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 90% identity to SEQ ID NO:29.In some embodiments, the amino acid sequence of the outer VL region of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 95% identity to SEQ ID NO: 30, and the amino acid sequence of the outer VH region of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 95% identity to SEQ ID NO: 29. In some embodiments, the amino acid sequence of the outer VL region of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 99% identity to SEQ ID NO: 30, and the amino acid sequence of the outer VH region of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 99% identity to SEQ ID NO: 29.

[0194] In some embodiments, a bispecific molecule that binds CD163 comprises a light chain variable (VL) region and a heavy chain variable (VH) region; the VL region comprises a light chain CDR1 (CDRL1) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:42, a light chain CDR2 (CDRL2) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:43, and a light chain CDR3 (CDRL3) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:44; and the amino acid sequences of the VL regions outside of CDRL1, CDRL2, and CDRL3 have an overall sequence identity of at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:30. the V gene usage of the VL region is a kappa light chain; the VH region comprises a heavy chain CDR1 (CDRH1) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:39, a heavy chain CDR2 (CDRH2) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:40, and a heavy chain CDR3 (CDRH3) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:41; the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:29; and the V gene usage of the VH region is an IGHV gene. In some embodiments, the amino acid sequence of the VL region outside of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 90% identity to SEQ ID NO:30, and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 90% identity to SEQ ID NO:29.In some embodiments, the amino acid sequence of the VL region outside of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 95% identity to SEQ ID NO: 30, and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 95% identity to SEQ ID NO: 29. In some embodiments, the amino acid sequence of the VL region outside of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 99% identity to SEQ ID NO: 30, and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 99% identity to SEQ ID NO: 29. In some embodiments, the V gene usage of the VL region is a kappa 8 light chain. In some embodiments, the V gene usage of the VL region is IGKV1D-39*01. In some embodiments, the V gene usage of the VH region is an IGHV4 gene. In some embodiments, the V gene usage of the VH region is IGHV4-b*01.

[0195] In some embodiments, the antigen-binding region specific for CD163 comprises the VH chain of SEQ ID NO:31: [ka] The CDRs are shown in bold and underlined and include amino acids 31-35 (CDR1), 50-65 (CDR2), and 99-122 (CDR3).

[0196] In some embodiments, the antigen-binding region specific for CD163 comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:31.

[0197] In some embodiments, the antigen binding region specific for CD163 comprises a VH region comprising one or more CDR sequences selected from the following: SYAMH (SEQ ID NO:45) VISYDGSNKYYADSVK (SEQ ID NO:46) ENVRPYYDFWSGYYSEYYYYGMDV (sequence number 47).

[0198] In some embodiments, the antigen-binding region specific for CD163 comprises the VL chain of SEQ ID NO: 32: [ka] The CDRs are shown in bold and underlined and include amino acids 24-34 (CDR1), 50-55 (CDR2), and 89-98 (CDR3).

[0199] In some embodiments, the antigen-binding region specific for CD163 comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:32.

[0200] In some embodiments, the antigen binding region specific for CD163 comprises a VL region comprising one or more CDR sequences selected from the following: RASQSISSYLN (SEQ ID NO: 48) AASSLQS (SEQ ID NO:49) QQSYSTPRGT (sequence number 50).

[0201] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence (SEQ ID NO:31).

[0202] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having at least 90% identity to the amino acid sequence (SEQ ID NO:31).

[0203] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having at least 95% identity to the amino acid sequence (SEQ ID NO:31).

[0204] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having at least 99% identity to the amino acid sequence (SEQ ID NO:31).

[0205] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a heavy chain variable region (VH) having 100% identity to the amino acid sequence (SEQ ID NO:31). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having at least 80% identity to the amino acid sequence (SEQ ID NO:32). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having at least 90% identity to the amino acid sequence (SEQ ID NO:32). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having at least 95% identity to the amino acid sequence (SEQ ID NO:32). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having at least 99% identity to the amino acid sequence (SEQ ID NO:32). In some embodiments, the bispecific molecule further comprises a light chain variable region (VL) having 100% identity to the amino acid sequence (SEQ ID NO:32).

[0206] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having at least 80% identity to the amino acid sequence (SEQ ID NO:32).

[0207] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having at least 90% identity to the amino acid sequence (SEQ ID NO:32).

[0208] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having at least 95% identity to the amino acid sequence (SEQ ID NO:32).

[0209] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having at least 99% identity to the amino acid sequence (SEQ ID NO:32).

[0210] In some embodiments, described herein are bispecific molecules comprising an antigen-binding region comprising a light chain variable region (VL) having 100% identity to the amino acid sequence (SEQ ID NO:32). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence (SEQ ID NO:31). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having at least 90% identity to the amino acid sequence (SEQ ID NO:31). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having at least 95% identity to the amino acid sequence (SEQ ID NO:31). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having at least 99% identity to the amino acid sequence (SEQ ID NO:31). In some embodiments, the bispecific molecule further comprises a heavy chain variable region (VH) having 100% identity to the amino acid sequence (SEQ ID NO:31).

[0211] In some embodiments, described herein are bispecific molecules comprising a heavy chain variable region (VH) having at least 80% identity to the amino acid sequence (SEQ ID NO:31) and a light chain variable region (VL) having at least 80% identity to the amino acid sequence (SEQ ID NO:32). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having at least 85% identity to the amino acid sequence (SEQ ID NO:32). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having at least 90% identity to the amino acid sequence (SEQ ID NO:32). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having at least 95% identity to the amino acid sequence (SEQ ID NO:32). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having at least 99% identity to the amino acid sequence (SEQ ID NO:32). In some embodiments, the bispecific molecule comprises a light chain variable region (VL) having 100% identity to the amino acid sequence (SEQ ID NO:32). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 85% identity to the amino acid sequence (SEQ ID NO:31). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 90% identity to the amino acid sequence (SEQ ID NO:31). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 95% identity to the amino acid sequence (SEQ ID NO:31). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 99% identity to the amino acid sequence (SEQ ID NO:31). In some embodiments, the bispecific molecule comprises a heavy chain variable region (VH) having at least 100% identity to the amino acid sequence (SEQ ID NO:31).

[0212] In some embodiments, described herein are bispecific molecules comprising a heavy chain sequence comprising a complementarity determining region (CDR) H1 having at least 80% identity to the amino acid sequence (SEQ ID NO:45), a CDRH2 having at least 80% identity to the amino acid sequence (SEQ ID NO:46), and a CDRH3 having at least 80% identity to the amino acid sequence (SEQ ID NO:47), and a light chain sequence comprising a CDRL1 having at least 80% identity to the amino acid sequence (SEQ ID NO:48), a CDRL2 having at least 80% identity to the amino acid sequence (SEQ ID NO:49), and a CDRL3 having at least 80% identity to the amino acid sequence (SEQ ID NO:50). In some embodiments, the bispecific molecules comprise a light chain sequence comprising a CDRL1 having at least 85% identity to the amino acid sequence (SEQ ID NO:48), a CDRL2 having at least 85% identity to the amino acid sequence (SEQ ID NO:49), and a CDRL3 having at least 85% identity to the amino acid sequence (SEQ ID NO:50). In some embodiments, the bispecific molecule comprises a light chain sequence comprising a CDRL1 having at least 90% identity to the amino acid sequence (SEQ ID NO:48), a CDRL2 having at least 90% identity to the amino acid sequence (SEQ ID NO:49), and a CDRL3 having at least 90% identity to the amino acid sequence (SEQ ID NO:50). In some embodiments, the bispecific molecule comprises a light chain sequence comprising a CDRL1 having at least 95% identity to the amino acid sequence (SEQ ID NO:48), a CDRL2 having at least 95% identity to the amino acid sequence (SEQ ID NO:49), and a CDRL3 having at least 95% identity to the amino acid sequence (SEQ ID NO:50). In some embodiments, the bispecific molecule comprises a light chain sequence comprising a CDRL1 having at least 99% identity to the amino acid sequence (SEQ ID NO:48), a CDRL2 having at least 99% identity to the amino acid sequence (SEQ ID NO:49), and a CDRL3 having at least 99% identity to the amino acid sequence (SEQ ID NO:50). In some embodiments, the bispecific molecule comprises a light chain sequence comprising a CDRL1 having 100% identity to the amino acid sequence (SEQ ID NO:48), a CDRL2 having 100% identity to the amino acid sequence (SEQ ID NO:49), and a CDRL3 having at least 100% identity to the amino acid sequence (SEQ ID NO:50).In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 85% identity to the amino acid sequence (SEQ ID NO:45), a CDRH2 having at least 85% identity to the amino acid sequence (SEQ ID NO:46), and a CDRH3 having at least 85% identity to the amino acid sequence (SEQ ID NO:47). In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 90% identity to the amino acid sequence (SEQ ID NO:45), a CDRH2 having at least 90% identity to the amino acid sequence (SEQ ID NO:46), and a CDRH3 having at least 90% identity to the amino acid sequence (SEQ ID NO:47). In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 95% identity to the amino acid sequence (SEQ ID NO:45), a CDRH2 having at least 95% identity to the amino acid sequence (SEQ ID NO:46), and a CDRH3 having at least 95% identity to the amino acid sequence (SEQ ID NO:47). In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 99% identity to the amino acid sequence (SEQ ID NO: 45), a CDRH2 having at least 99% identity to the amino acid sequence (SEQ ID NO: 46), and a CDRH3 having at least 99% identity to the amino acid sequence (SEQ ID NO: 47). In some embodiments, the bispecific molecule comprises a heavy chain sequence comprising a CDRH1 having at least 100% identity to the amino acid sequence (SEQ ID NO: 45), a CDRH2 having at least 100% identity to the amino acid sequence (SEQ ID NO: 46), and a CDRH3 having at least 100% identity to the amino acid sequence (SEQ ID NO: 47).

[0213] In some embodiments, a light chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:48; Described herein are bispecific molecules that bind to CD163, comprising at least one of: a light chain CDR2 having an amino acid sequence that is 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; and a light chain CDR3 having an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:50. In some embodiments, a bispecific molecule that binds to CD163 comprises at least one of: a light chain CDR1 having 100% identity to the amino acid sequence set forth as SEQ ID NO: 48; a light chain CDR2 having 100% identity to the amino acid sequence set forth as SEQ ID NO: 49; and a light chain CDR3 having 100% identity to the amino acid sequence set forth as SEQ ID NO: 50.

[0214] In some embodiments, a heavy chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:45; Described herein are bispecific molecules that bind to CD163, comprising at least one of: a heavy chain CDR2 having an amino acid sequence that is 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; and a heavy chain CDR3 having an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:47. In some embodiments, a bispecific molecule that binds to CD163 comprises at least one of: a heavy chain CDR1 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 45; a heavy chain CDR2 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 46; and a heavy chain CDR3 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 47.

[0215] In some embodiments, a light chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:48; a light chain CDR2 having an amino acid sequence that is 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:50; a light chain CDR3 having an amino acid sequence that is at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:45; a heavy chain CDR1 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:46; , 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO:47; and a heavy chain CDR3 having an amino acid sequence at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth as SEQ ID NO:47.In some embodiments, a bispecific molecule that binds to CD163 comprises at least one of: a light chain CDR1 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 48; a light chain CDR2 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 49; a light chain CDR3 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 50; a heavy chain CDR1 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 45; a heavy chain CDR2 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 46; and a heavy chain CDR3 having 100% identical to the amino acid sequence set forth as SEQ ID NO: 47.

[0216] In some embodiments, bispecific molecules are described herein that comprise a VL chain and a VH chain that confer binding specificity to CD163, in which amino acids within the framework can be altered, hi some embodiments, the amino acid changes comprise the introduction of conservative amino acid substitutions. In some embodiments, a bispecific molecule that binds CD163 comprises a light chain variable (VL) region and a heavy chain variable (VH) region; the VL region comprises a light chain CDR1 (CDRL1) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:48, a light chain CDR2 (CDRL2) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:49, and a light chain CDR3 (CDRL3) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:50; and the amino acid sequences of the VL regions outside of CDRL1, CDRL2, and CDRL3 are at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical to SEQ ID NO:32. , 98%, or 99% identity; the VH region comprises a heavy chain CDR1 (CDRH1) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:45, a heavy chain CDR2 (CDRH2) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:46, and a heavy chain CDR3 (CDRH3) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:47; and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:31. In some embodiments, the amino acid sequence of the VL region outside of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 90% identity to SEQ ID NO:32, and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 90% identity to SEQ ID NO:31.In some embodiments, the amino acid sequence of the outer VL region of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 95% identity to SEQ ID NO: 32, and the amino acid sequence of the outer VH region of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 95% identity to SEQ ID NO: 31. In some embodiments, the amino acid sequence of the outer VL region of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 99% identity to SEQ ID NO: 32, and the amino acid sequence of the outer VH region of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 99% identity to SEQ ID NO: 31.

[0217] In some embodiments, a bispecific molecule that binds CD163 comprises a light chain variable (VL) region and a heavy chain variable (VH) region; the VL region comprises a light chain CDR1 (CDRL1) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:48, a light chain CDR2 (CDRL2) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:49, and a light chain CDR3 (CDRL3) having an amino acid sequence that is 100% identical to the amino acid sequence set forth as SEQ ID NO:50; and the amino acid sequences of the VL regions outside of CDRL1, CDRL2, and CDRL3 have an overall sequence identity of at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:32. the V gene usage of the VL region is a kappa light chain; the VH region comprises a heavy chain CDR1 (CDRH1) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:45, a heavy chain CDR2 (CDRH2) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:46, and a heavy chain CDR3 (CDRH3) having an amino acid sequence 100% identical to the amino acid sequence set forth as SEQ ID NO:47; the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:31; and the V gene usage of the VH region is an IGHV gene. In some embodiments, the amino acid sequence of the VL region outside of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 90% identity to SEQ ID NO:32, and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 90% identity to SEQ ID NO:31.In some embodiments, the amino acid sequence of the VL region outside of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 95% identity to SEQ ID NO: 32, and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 95% identity to SEQ ID NO: 31. In some embodiments, the amino acid sequence of the VL region outside of CDRL1, CDRL2, and CDRL3 has an overall sequence identity of at least 99% identity to SEQ ID NO: 32, and the amino acid sequence of the VH region outside of CDRH1, CDRH2, and CDRH3 has an overall sequence identity of at least 99% identity to SEQ ID NO: 31. In some embodiments, the V gene usage of the VL region is VK1.O12. In some embodiments, the V gene usage of the VH region is an IGHV3 gene. In some embodiments, the V gene usage of the VH region is IGHV3.30-3.

[0218] Treatment In certain aspects, the subject matter described herein relates to a method for treating or preventing cancer in a subject suffering from cancer. In some embodiments, the method comprises administering to a subject a bispecific molecule comprising at least two antigen-binding regions, each antigen-binding region binding to a different antigen on a first cell (TFC) of the cancer. In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising any one of the bispecific molecules described herein.

[0219] In some embodiments, the first antigen is an epithelial cell lineage marker. In some embodiments, the epithelial marker is any one of the markers in FIG. 2 and the epithelial cell lineage marker is any one of the markers in FIG. 2. In some embodiments, the epithelial cell lineage marker is epithelial cell adhesion molecule (EpCAM). In some embodiments, the second antigen is a macrophage cell lineage marker. In some embodiments, the macrophage marker is any one of the markers in FIG. 1. In some embodiments, the macrophage cell lineage marker is CD163. In some embodiments, both antigens are macrophage cell lineage markers. In some embodiments, the first antigen is CD117, CD34, CD123, or any combination thereof, and the second antigen is CD163. In some embodiments, the bispecific antibody, functional equivalent thereof, antigen-binding fragment thereof, derivative thereof, or antibody-like bispecific molecule binds to two different antigens selected from FIG. 1.

[0220] In some embodiments, the bispecific molecule is a bispecific antibody. In some embodiments, the bispecific antibody is conjugated to a drug. In some embodiments, the drug is a toxin. In some embodiments, the drug is a chemotherapeutic agent. In some embodiments, the bispecific molecule comprises a dual nanobody, a BiTE, a tandAb, a DART, a DART-Fc, a DARPin, a scFv, a scFv-HAS-scFV, and a DNL-Fab3. In some embodiments, the bispecific molecule is a bispecific chimeric antigen receptor (CAR). In some embodiments, the bispecific molecule is a split CAR-T. In some embodiments, the bispecific molecule is a Co-LOCKR comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer. In some embodiments, the bispecific CAR is a synNotch CAR. In some embodiments, the bispecific CAR binds to a Co-LOCKR that comprises a first polypeptide, a second polypeptide, and a third polypeptide, where the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer, and the third polypeptide binds to the bispecific CAR, where the third polypeptide is operably linked to the first polypeptide or the second polypeptide.

[0221] In some embodiments, the present invention provides a method comprising administering to a subject a pharmaceutical composition comprising any one of the bispecific molecules described herein, wherein a first antigen-binding region binds to EpCAM and a second antigen-binding region binds to CD163. Antigen-binding regions specific for EpCAM are described herein, including but not limited to, in the section entitled "Antigen-binding regions or domains specific for EpCAM". Antigen-binding regions specific for CD163 are described herein, including but not limited to, in the section entitled "Antigen-binding regions or domains specific for CD163". The treatment method of the present invention comprises using any of the bispecific molecules described herein of an antigen-binding region specific for EpCAM in combination with any of the antigen-binding regions specific for CD163.

[0222] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is breast cancer, brain cancer, gastrointestinal cancer including stomach cancer and colon cancer, pancreatic cancer, renal cancer, liver cancer, lung cancer, thymic cancer, ovarian cancer, prostate cancer, or endometrial cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the liquid cancer is a leukemia, lymphoma, or myeloma. In some embodiments, the liquid cancer is a B cell malignancy. In some embodiments, the B cell malignancy is multiple myeloma. In some embodiments, the B cell malignancy is a B cell lymphoma. In some embodiments, the B cell malignancy is diffuse large B cell lymphoma (DLBCL). In some embodiments, the B cell malignancy is non-Hodgkin's lymphoma (NHL). In some embodiments, the B cell malignancy is chronic lymphocytic leukemia (CLL). In some embodiments, the liquid cancer is acute myeloid leukemia (AML). In some embodiments, the liquid cancer is a myeloid tumor. In some embodiments, the liquid cancer is myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS / MPN overlap syndrome, acute myeloid leukemia, or chronic myeloid leukemia. In some embodiments, the TFC is a metastatic TFC.

[0223] Myeloid leukemia In some embodiments, the cancer is myeloid leukemia. In some embodiments, the myeloid leukemia TFC expresses a first antigen and a second antigen. In some embodiments, the first antigen and the second antigen are both macrophage cell lineage markers. In some embodiments, the first antigen and the second antigen are any one pair of markers in FIG. 1. In some embodiments, the first antigen is CD117, CD34, CD123, or a combination thereof. In some embodiments, the second antigen is CD163. In some embodiments, the bispecific molecules disclosed herein bind to two antigens, both of which are macrophage cell lineage markers.

[0224] Myeloid tumors In some embodiments, the cancer is a myeloid tumor. In some embodiments, the myeloid tumor is a myelodysplastic syndrome (MDS). In some embodiments, the myeloid tumor is a myeloproliferative neoplasm (MPN). In some embodiments, the myeloid tumor is an overlapping MDS / MPN syndrome. In some embodiments, the myeloid tumor is acute or chronic myeloid leukemia.

[0225] Cancer Treatment There are many cancer treatment options available today. Surgery is performed to remove the cancer or tumor mass. Chemotherapy involves administering toxic drugs to the patient to kill the cancer cells. Radiation therapy utilizes high-powered energy beams, such as x-rays or protons, to kill the cancer cells. Bone marrow can be transplanted from a healthy individual into a cancer patient to replace the patient's own diseased bone marrow. Bone marrow produces blood cells from blood stem cells, and bone marrow transplants are performed for the purpose of treating or curing liquid cancer. Bone marrow transplants also allow for the use of high doses of chemotherapy to treat cancer patients. Immunotherapy utilizes the body's own immune system to fight cancer. Immunotherapy can help the patient's immune system recognize and attack the cancer cells. Hormonal therapy can be used to treat breast or prostate cancer, which can be supported by hormones in the body. Blocking their effects on the body can stop the cancer from growing. Cryoablation kills cancer cells by reducing the temperature using thin needles (cryoprobes) inserted directly into the cancer tumor through the patient's skin. Radiofrequency ablation uses electrical energy to heat and thereby kill cancer cells. Radiofrequency energy is directed through a needle to heat the surrounding tissue. Each of these procedures can be administered in combination with the methods described herein.

[0226] Diagnostic methods In certain aspects, the subject matter described herein relates to a method of diagnosing cancer. In some embodiments, the method comprises detecting cells expressing at least one epithelial cell lineage marker and at least one macrophage cell lineage marker. In some embodiments, the at least one epithelial cell lineage marker is any one of the markers in FIG. 2. In some embodiments, the at least one epithelial cell lineage marker is epithelial cell adhesion molecule (EpCAM). In some embodiments, the at least one marker of macrophage cell lineage is any one of the markers in FIG. 1. In some embodiments, the at least one marker of macrophage cell lineage is CD163. In some embodiments, the method comprises detecting cells expressing at least two macrophage cell lineage markers. In some embodiments, the first marker is CD117, CD34, CD123, or any combination thereof, and the second marker is CD163. In some embodiments, the at least two macrophage cell lineage markers are any two of the markers in FIG. 1. In some embodiments, the detection comprises an assay in which the bispecific molecule binds to at least one epithelial cell lineage marker and at least one macrophage cell lineage marker. In some embodiments, the detection comprises an assay in which the bispecific molecule binds to at least two macrophage cell lineage markers. In some embodiments, the detection comprises a flow cytometry assay. In some embodiments, the detection comprises an immunostaining assay. In some embodiments, the antibodies binding to each antigen are each conjugated to a different fluorescent dye, and the fluorescent dyes are detected using either flow cytometry or immunostaining. In some embodiments, the immunostaining is immunofluorescence staining. In some embodiments, the detection comprises a step of size-separating the cells prior to the assay, as shown in FIG. 4. In some embodiments, larger cells are selected and assayed during size separation.

[0227] In some embodiments, the bispecific molecule is a bispecific antibody. In some embodiments, the bispecific antibody is conjugated to a drug. In some embodiments, the drug is a toxin. In some embodiments, the drug is a chemotherapeutic agent. In some embodiments, the bispecific molecule comprises a dual nanobody, a BiTE, a tandAb, a DART, a DART-Fc, a DARPin, a scFv, a scFv-HAS-scFV, and a DNL-Fab3. In some embodiments, the bispecific molecule is a bispecific chimeric antigen receptor (CAR). In some embodiments, the bispecific molecule is a Co-LOCKR comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer. In some embodiments, the bispecific CAR is a synNotch CAR. In some embodiments, the bispecific CAR binds to a Co-LOCKR that comprises a first polypeptide, a second polypeptide, and a third polypeptide, where the first polypeptide and the second polypeptide each bind to a different antigen on the TFC of the cancer, and the third polypeptide binds to the bispecific CAR, where the third polypeptide is operably linked to the first polypeptide or the second polypeptide.

[0228] In some embodiments, the invention provides a method comprising detecting cells expressing at least one epithelial cell lineage marker and at least one macrophage cell lineage marker using any one of the bispecific molecules described herein, where a first antigen-binding region binds EpCAM and a second antigen-binding region binds CD163. Antigen-binding regions specific for EpCAM are described herein, including but not limited to, in the section entitled "Antigen-binding Regions or Domains Specific for EpCAM". Antigen-binding regions specific for CD163 are described herein, including but not limited to, in the section entitled "Antigen-binding Regions or Domains Specific for CD163". Diagnostic methods of the invention include using any of the bispecific molecules described herein of an antigen-binding region specific for EpCAM in combination with any of the antigen-binding regions specific for CD163.

[0229] Pharmaceutical Compositions In certain aspects, pharmaceutical compositions comprising the bispecific molecules described above are also provided. In some embodiments, the subject matter described herein relates to pharmaceutical compositions comprising an effective amount of a bispecific molecule described herein and a pharma- ceutical acceptable diluent, carrier, or excipient.

[0230] As used herein, "pharmaceutical composition" refers to a therapeutically effective formulation according to the present invention. As used herein, "therapeutically effective amount" or "effective amount" or "therapeutically effective" refers to an amount that provides a therapeutic effect for a given condition and administration regimen. Therapeutically effective amounts can be determined by one of skill in the art based on patient characteristics, such as age, weight, sex, condition, comorbidities, other diseases, etc., as is well known in the art.

[0231] In some embodiments, the pharmaceutical compositions described herein can be administered as solid compositions. In some embodiments, the solid compositions include excipients including, but not limited to, lactose, starch, cellulose, milk sugar, or high molecular weight polyethylene glycols. In some embodiments, the pharmaceutical compositions described herein can be administered as aqueous suspensions and / or elixirs. In some embodiments, the pharmaceutical compositions described herein can be combined with various sweeteners or flavorings, colorants or dyes, emulsifiers and / or suspending agents, diluents (e.g., water, ethanol, propylene glycol, and glycerin), and combinations thereof.

[0232] In some embodiments, the pharmaceutical compositions described herein can be administered parenterally, for example, intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly, or subcutaneously, or by infusion techniques.In some embodiments, the pharmaceutical compositions described herein can be administered in the form of a sterile aqueous solution, which may contain other substances, for example, sufficient salts or glucose to make it isotonic with blood.The preparation of suitable parenteral formulations under sterile conditions can be easily accomplished by standard pharmaceutical techniques known to those skilled in the art.

[0233] In some embodiments, pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bactericides, and solutes (to render the formulation isotonic with the blood of the intended recipient) and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. The pharmaceutical compositions may be presented in unit-dose or multi-dose containers. The pharmaceutical compositions may be in sealed ampoules or vials. The pharmaceutical compositions may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use.

[0234] Polynucleotides In certain aspects, polynucleotides encoding the bispecific molecules or portions thereof described herein are also provided. In some embodiments, polynucleotides encoding the bispecific molecules or portions thereof described herein are isolated from cells expressing the bispecific molecules or portions thereof described herein according to methods available in the art, including amplification by polymerase chain reaction. One or more polynucleotides encoding one or more bispecific molecules or portions thereof described herein can be subcloned into one or more expression vectors. In some embodiments, expression vectors comprising polynucleotides encoding bispecific molecules or portions thereof can be used to recombinantly produce the bispecific molecules or portions thereof described herein using procedures known in the art.

[0235] vector In certain aspects, vectors are also provided that include one or more polynucleotides encoding one or more bispecific molecules or portions thereof described herein. In some embodiments, the bispecific molecules or portions thereof described herein are recombinantly produced using any suitable vector and method known in the art. Suitable expression vectors include, but are not limited to, pcDNA3.4, pcDNA3.3 Topo, pOptiVec, pSG5L, pDEST27, pCI, pIRES, pBApo, pSF-CMV, and pEF4 / V5HisA.

[0236] virus In certain aspects, viruses are also provided that contain polynucleotides encoding bispecific molecules. Suitable virus-based protein expression systems are known in the art, including, but not limited to, lentivirus and adenovirus expression systems. Suitable lentivirus vectors include, but are not limited to, pHIV-dTomato, pAWp11, LRG2.1, LT3, LentiV, pCCL, pCS, and pHIV-eGFP. Suitable adenovirus vectors include, but are not limited to, pAd / CMV / V5-DEST, pAdenoX, pICPIS, pAAV, and pAd / PL-DEST. Methods for protein expression using virus-based expression systems are well known in the art.

[0237] cell In certain embodiments, genetically modified, transformed or transduced host cells comprising the bispecific molecules and / or polynucleotides encoding the bispecific molecules are also provided. Suitable cells known in the art include, but are not limited to, T cells, HL-60 cells, CHO cells, HEK293 cells, 293T cells, E. coli, DH5a. These cells can be genetically modified, transformed or transduced with the bispecific molecules and / or polynucleotides encoding the bispecific molecules according to any method known in the art. References: 1 Borot, F., Mukherjee, S. & Ali, AMChallenges and Solutions to Bringing Chimeric Antigen Receptor T-Cell Therapy to Myeloid Malignancies.Cancer J 27,143-150,doi:10.1097 / PPO.0000000000000512(2021). 2 Borot,F.et al.Gene-edited stem cells enable CD33-directed immune therapy for myeloid malignancies.Proc Natl Acad Sci U S A 116,11978-11987,doi:10.1073 / pnas.1819992116(2019). 3 Wang,H.F.et al.Cell fusion in cancer hallmarks: Current research status and future indications.Oncol Lett 22,530,doi:10.3892 / ol.2021.12791(2021). 4 Vignery,A.Macrophage fusion: are somatic and cancer cells possible partners? Trends Cell Biol 15,188-193,doi:10.1016 / j.tcb.2005.02.008(2005). 5 Sutton,T.L.,Walker,B.S.& Wong,M.H.Circulating Hybrid Cells Join the Fray of Circulating Cellular Biomarkers.Cell Mol Gastroenterol Hepatol 8,595-607,doi:10.1016 / j.jcmgh.2019.07.002(2019). 6 Shabo,I.et al.Macrophage traits in cancer cells are induced by macrophage-cancer cell fusion and cannot be explained by cellular interaction.BMC Cancer 15,922,doi:10.1186 / s12885-015-1935-0(2015). 7 Adams,D.L.et al.Circulating giant macrophages as a potential biomarker of solid tumors.Proc Natl Acad Sci U S A 111,3514-3519,doi:10.1073 / pnas.1320198111(2014). 8 Shabo,I.,Olsson,H.,Sun,X.F.& Svanvik,J.Expression of the macrophage antigen CD163 in rectal cancer cells is associated with early local recurrence and reduced survival time.Int J Cancer 125,1826-1831,doi:10.1002 / ijc.24506(2009). 9 Aljabery,F.,Olsson,H.,Gimm,O.,Jahnson,S.& Shabo,I.M2-macrophage infiltration and macrophage traits of tumor cells in urinary bladder cancer.Urol Oncol 36,159 e119-159 e126,doi:10.1016 / j.urolonc.2017.11.020(2018). 10 Sodi,S.A.et al.Melanoma x macrophage fusion hybrids acquire increased melanogenesis and metastatic potential: altered N-glycosylation as an underlying mechanism.Pigment Cell Res 11,299-309,doi:10.1111 / j.1600-0749.1998.tb00739.x(1998). 11 Rachkovsky,M.et al.Melanoma x macrophage hybrids with enhanced metastatic potential.Clin Exp Metastasis 16,299-312,doi:10.1023 / a:1006557228604(1998). 12 Dittmar,T.et al.Recurrence cancer stem cells--made by cell fusion? Med Hypotheses 73,542-547,doi:10.1016 / j.mehy.2009.05.044(2009). 13 Clawson,G.A.et al.“Stealth dissemination”of macrophage-tumor cell fusions cultured from blood of patients with pancreatic ductal adenocarcinoma.PLoS One 12,e0184451,doi:10.1371 / journal.pone.0184451(2017). 14 Aguirre,L.A.et al.Tumor stem cells fuse with monocytes to form highly invasive tumor-hybrid cells.Oncoimmunology 9,1773204,doi:10.1080 / 2162402X.2020.1773204(2020). 15 Lajoie,M.J.et al.Designed protein logic to target cells with precise combinations of surface antigens.Science 369,1637-1643,doi:10.1126 / science.aba6527(2020). 16 Roybal,K.T.et al.Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors.Cell 167,419-432 e416,doi:10.1016 / j.cell.2016.09.011(2016). 17 Morsut,L.et al.Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors.Cell 164,780-791,doi:10.1016 / j.cell.2016.01.012(2016). 18 Kloss,C.C.,Condomines,M.,Cartellieri,M.,Bachmann,M.& Sadelain,M.Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells.Nat Biotechnol 31,71-75,doi:10.1038 / nbt.2459(2013). 19 Lee,S.& Wong,W.W.The Most Logical Approach to Improve CAR T Cell Therapy.Cell Syst 11,421-423,doi:10.1016 / j.cels.2020.10.008(2020). 20 Cho,J.H.,Collins,J.J.& Wong,W.W.Universal Chimeric Antigen Receptors for Multiplexed and Logical Control of T Cell Responses.Cell 173,1426-1438 e1411,doi:10.1016 / j.cell.2018.03.038(2018). 21 Kittel-Boselli, E. et al. Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy. Cancers(Basel)13, doi:10.3390 / cancers13194785(2021). 22 Feldmann, A. et al.Versatile chimeric antigen receptor platform for controllable and combinatorial T cell therapy.Oncoimmunology 9,1785608,doi:10.1080 / 2162402X.2020.1785608(2020). 23 Salzer,B.et al.Engineering AvidCARs for combinatorial antigen recognition and reversible control of CAR function.Nat Commun 11,4166,doi:10.1038 / s41467-020-17970-3(2020).

[0238] Example 1. Generation of cell line models for expression of lineage-specific antigens. We generated a cell line model that recapitulates the expression of two lineage-specific antigens (LSAs) on "first cells," also referred to herein as tumor-macrophage hybrid (TMH) target cells (Figure 5A-D).

[0239] LSA Selection: TMH cells are formed by the fusion of cells from two different lineages. The fusion results in a rearrangement of biomolecules, including chromatin, transcriptome, and proteome, which results in hybrid cells that retain the expression of specific antigens of both lineages (1-3). Theoretically, LSAs from either lineage should be present in the hybrid cells. Figures 1 and 2 show a list of CD antigens of epithelial or macrophage lineages. The expression of several LSAs on TMHs has been reported (4, 5). The LSAs selected based on the expression of these antigens on TMHs were EpCAM and CD163 (1, 2, 4, 6-19). The protein sequences of EpCAM or CD163 and their isoforms can be found in Table 1 below.

[0240] Cell line: HL-60 (CCL-240; ATCC) cell line is used to express LSA. HL-60 cells were cultured in IMDM medium supplemented with 10% fetal bovine serum (FBS) and 1X penicillin and streptomycin at 37°C and 5% CO2 in a humidified chamber.

[0241] Plasmid DNA: Plasmids encoding EpCAM (HsCD00954335; DNASU repository) and CD163 (HsCD00861066; DNASU repository) with a lentiviral backbone (pLenti6.3 / V5-DEST) and a C-terminal V5 tag were amplified in E. coli (DH5a) cells and plasmid DNA was purified using a Qiagen Maxi kit. The sequences of EpCAM or CD163 expressed in HL-60 cells are shown in Table 2.

[0242] Lentivirus: Lentiviral particles of EpCAM and CD163 expression plasmids were obtained by co-transfection with a helper plasmid (encoding VSVG coat protein) in 293 cells. 239 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1X penicillin and streptomycin at 37°C and 5% CO2 in a humidified chamber. Lentiviral particles from cell supernatants were purified using PEG and resuspended in IMDM medium.

[0243] Viral transduction and selection: Lentiviral particles were used to transduce HL-60 cells. Concentrated lentiviral particles were co-incubated with fibronectin-treated cell cultures and HL-60 cells. After 48 hours of co-culture, cells expressing EpCAM or CD163 or both were selected using the antibiotic blasticidin.

[0244] Phenotypic analysis: EpCAM and CD163 expression was measured using flow cytometry (Figure 6A-C). Cells were stained with a 1:100 dilution of fluorochrome-gated monoclonal antibodies recognizing human EpCAM (clone 9C4 conjugated to PerCP / Cyanine5.5, Biolegend 324213) or CD163 (clone RM3 / 1 conjugated to PE, Biolegend 326506) and data were acquired on a BD LSRII flow cytometer and Diva software. Flow cytometry data were analyzed using FlowJo (FlowJo LLC) or FCS Express (De NovoS oftware).

[0245] [Table 1-1]

[0246] [Table 1-2]

[0247] [Table 1-3]

[0248] [Table 1-4]

[0249] [Table 2]

[0250] Example 2. Generation of Co-LOCKR Protein To perform precise targeting of TMHs characterized by the expression of LSAs from two different lineages and to miss cells of the parental lineage (expressing a single LSA), we used a colocalization-dependent protein-based logic gate design that performs an AND Boolean logic operation to target TMHs with precise combinations of surface antigens (EpCAM or CD163) (20). A de novo protein switch that is activated upon colocalization to calculate AND logic has been described, which contains the latching orthogonal cage-key protein (Co-LOCKR). Co-LOCKR is composed of a cage and key protein that activates through a conformational change when the cage and key colocalize (Figure 7A-C) (20). The cage module is composed of a "latch" that sequesters a functional peptide (Bim) in an inactive conformation but can bind an effector (Bcl2) upon conformational change. The effector can be a protein-drug conjugate (Bcl2 conjugated to a cytotoxic compound) or chimeric antigen receptor T cells (CAR-T) expressing Bcl2CAR (20). Both the cage and the key are modular and can be coupled to an antigen-binding domain to recruit the cage and the key to cells expressing the target antigen (EpCAM or CD163). The antigen-binding domain can be a single chain variable fragment (ScFv) or Darpin, or any molecule with affinity for the target.

[0251] Design: The schematic design of cage and key targeting EpCAM and CD163 is shown in Figure 8A-B. A typical cage and key target protein contains a signal peptide from mouse immunoglobulin kappa (mIgk), a 6x histidine tag (His6) for protein purification, a tobacco etch virus (TEV) protease site for tag removal, a key or cage domain, and an antigen binding domain. Cage and key proteins were designed against both EpCAM and CD163 using both "wild type" sequences or variants of key and cage, resulting in a total of eight proteins (Table 3). For the antigen binding domain binding to EpCAM, we selected anti-EpCAM ScFv: the variable light (VL) and variable heavy (VH) sequences of anti-EpCAM ScFv are described in patent US7,632,925B2, the contents of which are incorporated herein by reference in their entirety. For the antigen binding domain that binds to CD163, the inventors selected anti-CD163 ScFv: the variable light chain (VL) and variable heavy chain (VH) sequences of anti-CD163 ScFv are described in patent US9,724,426B2, the contents of which are incorporated herein by reference in their entirety. For the antigen binding domain that binds to CD163, the variable light chain (VL) and variable heavy chain (VH) sequences of anti-CD163 ScFv are described in patent US11,034,770B2, the contents of which are incorporated herein by reference in their entirety and can also be used. The cage sequence and key sequence are described in: Lajoie, MJ et al., Science. 2020; 369 (6511): 1637-43 (the contents of which are incorporated herein by reference in their entirety).

[0252] Synthesis and cloning of cage and key proteins: Codon-optimized DNA sequences encoding the protein sequences in Table 2 were synthesized as fragments and cloned into the mammalian expression vector pcDNA3.4. The resulting plasmids were amplified in E. coli (DH5a) cells and the plasmid DNA was purified using the Qiagen Maxi kit.

[0253] Expression and purification of Co-LOCKR protein 293 cells were transfected according to the manufacturer's protocol with MIRUS BIO™ TRANSIT™-293 transfection reagent (MIR 2700 Mirus Bio). 24 hours prior to transfection, 293 cells were plated in 10 cm tissue culture treated dishes to achieve 80% confluence on the day of transfection. Maxi DNA of each Co-LOCKR plasmid was mixed with TRANSIT™-293 reagent and serum-free medium and incubated at room temperature for 25 minutes. After incubation, the mixture was added dropwise to the 293 cells. The cells were returned to the incubator for 72-96 hours. Finally, the cell culture supernatant was collected and centrifuged to separate isolated cells and supplemented with phenylmethylsulfonyl fluoride (PMSF) at a final concentration of 1 mM to inhibit serine protease activity during purification. To purify the Co-LOCKR protein, 10 μL of TALON® Metal Affinity Resin (Takara Bio USA, San Jose, California) per ml of supernatant was washed with PBST. The culture supernatant containing the Co-LOCKR protein was rotated overnight at 4° C. The next day, the sample was centrifuged at 500 g for 5 minutes at 4° C. and the supernatant was aspirated. The resin was resuspended in PBST, rotated for 10 minutes at 4° C., and centrifuged as before. This washing process was repeated a total of three times. Per 100 μL of resin, 300 μL of elution buffer, 150 mM imidazole in PBS, was added to the resin bed and the sample was rotated at 4° C. for at least 1 hour. The sample was centrifuged at 500 g for 5 minutes at 4° C., gently resuspended by pipetting, and then transferred to a MICRO BIO-SPIN™ chromatography column (Bio-Rad Laboratories, Hercules, California). The column was placed in a microcentrifuge tube and centrifuged at 10,000 g for 1 min at 4° C. The eluate from the TALON® resin was diluted to a final volume of 6 mL in PBS and added to a 30 kDa MWCO protein concentrator (ThermoFisher). The sample was centrifuged at 3,000 g for 15 min.The flow-through from the lower chamber of the concentrator was discarded and the concentrated sample was diluted with 5 mL of PBS and then centrifuged under the conditions described above. The concentrated samples containing the His-purified experimental constructs were collected and either used immediately for subsequent experiments or frozen at -20°C until use.

[0254] Staining and immunoblot analysis: Co-LOCKR protein eluate was mixed with 2X-Laemmli sample buffer (Bio-Rad) containing β-mercaptoethanol. The mixture was heated at 95°C for 10 min, and 25 μL of sample was separated on a Novex 4-20% Tris-glycine minigel (Invitrogen, Waltham, Massachusetts). To confirm expression and purity, the gel was stained with Coomassie G-250 stain (Figure 9A). The gel was washed with deionized water for 5 min, stained with Coomassie G-250 for 20 min, and then destained for 10 min. The gel was photographed using a LiCOR imaging system. For immunoblot analysis, proteins from the gel were transferred to a nylon membrane, blocked using 5% milk prepared in PBST, and incubated overnight with anti-His6 primary antibody in 0.5% milk prepared in PBST. After overnight incubation, the membrane is washed a total of four times with PBST. The membrane was probed with anti-mouse 800CW secondary antibody (LI-COR Biosciences, Lincoln, Nebraska) for 30 min at room temperature. The secondary antibody was discarded and the membrane was washed four times as described above. The membrane was analyzed on an Odyssey XF imaging system (LI-COR) using ImageStudio software (LI-COR) (Figure 9B).

[0255] [Table 3-1]

[0256] [Table 3-2]

[0257] [Table 3-3]

[0258] Example 3 - Generation of split CAR-T In the split chimeric antigen receptor T cell (split CAR-T) system, two modules, one with a CAR and the other with a chimeric costimulatory receptor (CCR), are expressed on the same T cell to achieve balance of signaling and maximize the cytotoxic activity of T cells on target cells expressing two different target antigens (Figure 10) (21). The CAR module contains an antigen-binding domain that targets one antigen and a CD3z signaling domain. The CCR module contains an antigen-binding domain that targets another antigen and two or more costimulatory domains.

[0259] Design: The schematic design of the EpCAM and CD163 targeting CAR and CCR modules is shown in Figure 11. In some embodiments, the CAR module was designed to include 1) a signal peptide for membrane targeting (from GM-CSF or CD8 alpha), 2) an ScFv sequence (from either an anti-EpCAM or anti-CD163 antibody), 3) a hinge region (from CD8), 4) a transmembrane domain (from CD8), and 5) a CD3z signaling domain. In some embodiments, the CCR module was designed to include 1) a signal peptide for membrane targeting (from GM-CSF), 2) an ScFv sequence (from either an anti-EpCAM or anti-CD163 antibody), 3) a hinge region (from CD28), 4) a transmembrane domain (from CD28), and 5) a CD28 costimulatory domain, and 6) a 4-1BB costimulatory domain. For the antigen-binding domain binding to EpCAM, the inventors selected the anti-EpCAM ScFv: the variable light (VL) and variable heavy (VH) sequences of the anti-EpCAM ScFv are described in patent US7,632,925B2, the contents of which are incorporated herein by reference in their entirety. For the antigen-binding domain binding to CD163, the inventors selected the anti-CD163 ScFv: the variable light (VL) and variable heavy (VH) sequences of the anti-CD163 ScFv are described in patents US9,724,426B2 and US11,034,770B2, the contents of each of which are incorporated herein by reference in their entirety. The sequences of the various split CAR modules are listed in Table 4.

[0260] Synthesis and cloning of split CAR modules: Codon-optimized DNA sequences encoding the protein sequences in Table 4 were synthesized as fragments and cloned into the pHIV-dTomato or pHIV-eGFP lentiviral vector backbone. The resulting plasmids were amplified in E. coli (DH5a) cells and the plasmid DNA was purified using the Qiagen Maxi kit.

[0261] [Table 4-1]

[0262]

Table 4-2

[0263] References for Examples 1 to 3: 1. Gast CE,Silk AD,Zarour L,Riegler L,Burkhart JG,Gustafson KT,et al.Cell fusion potentiates tumor heterogeneity and reveals circulating hybrid cells that correlate with stage and survival.Sci Adv.2018;4(9):eaat7828. 2. Powell AE,Anderson EC,Davies PS,Silk AD,Pelz C,Impey S,et al.Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.Cancer Res.2011;71(4):1497-505. 3. Sutton TL,Walker BS,Wong MH.Circulating Hybrid Cells Join the Fray of Circulating Cellular Biomarkers.Cell Mol Gastroenterol Hepatol.2019;8(4):595-607. 4. Adams DL,Martin SS,Alpaugh RK,Charpentier M,Tsai S,Bergan RC,et al.Circulating giant macrophages as a potential biomarker of solid tumors.Proc Natl Acad Sci U S A.2014;111(9):3514-9. 5. Tang CM,Zhu P,Li S,Makarova OV,Amstutz PT,Adams DL.Blood-based biopsies-clinical utility beyond circulating tumor cells.Cytometry A.2018;93(12):1246-50. 6. Adams DL,Adams DK,Alpaugh RK,Cristofanilli M,Martin SS,Chumsri S,et al.Circulating Cancer-Associated Macrophage-Like Cells Differentiate Malignant Breast Cancer and Benign Breast Conditions.Cancer Epidemiol Biomarkers Prev.2016;25(7):1037-42. 7. Aguirre LA,Montalban-Hernandez K,Avendano-Ortiz J,Marin E,Lozano R,Toledano V,et al.Tumor stem cells fuse with monocytes to form highly invasive tumor-hybrid cells.Oncoimmunology.2020;9(1):1773204. 8. Aljabery F,Olsson H,Gimm O,Jahnson S,Shabo I.M2-macrophage infiltration and macrophage traits of tumor cells in urinary bladder cancer.Urol Oncol.2018;36(4):159 e19- e26. 9. Augustyn A,Adams DL,He J,Qiao Y,Verma V,Liao Z,et al.Giant Circulating Cancer-Associated Macrophage-Like Cells Are Associated With Disease Recurrence and Survival in Non-Small-Cell Lung Cancer Treated With Chemoradiation and Atezolizumab.Clin Lung Cancer.2021;22(3):e451-e65. 10. Clawson GA,Matters GL,Xin P,Imamura-Kawasawa Y,Du Z,Thiboutot DM,et al.Macrophage-tumor cell fusions from peripheral blood of melanoma patients.PLoS One.2015;10(8):e0134320. 11. Clawson GA,Matters GL,Xin P,McGovern C,Wafula E,dePamphilis C,et al.“Stealth dissemination”of macrophage-tumor cell fusions cultured from blood of patients with pancreatic ductal adenocarcinoma.PLoS One.2017;12(9):e0184451. 12. Pawelek JM.Tumour-cell fusion as a source of myeloid traits in cancer.Lancet Oncol.2005;6(12):988-93. 13. Pawelek JM.Fusion of bone marrow-derived cells with cancer cells: metastasis as a secondary disease in cancer.Chin J Cancer.2014;33(3):133-9. 14. Shabo I,Olsson H,Sun XF,Svanvik J.Expression of the macrophage antigen CD163 in rectal cancer cells is associated with early local recurrence and reduced survival time.Int J Cancer.2009;125(8):1826-31. 15. Shabo I,Stal O,Olsson H,Dore S,Svanvik J.Breast cancer expression of CD163,a macrophage scavenger receptor,is related to early distant recurrence and reduced patient survival.Int J Cancer.2008;123(4):780-6. 16. Shabo I,Svanvik J.Expression of macrophage antigens by tumor cells.Adv Exp Med Biol.2011;714:141-50. 17. Shabo I,Svanvik J,Lindstrom A,Lechertier T,Trabulo S,Hulit J,et al.Roles of cell fusion,hybridization and polyploid cell formation in cancer metastasis.World J Clin Oncol.2020;11(3):121-35. 18. Sieler M,Weiler J,Dittmar T.Cell-Cell Fusion and the Roads to Novel Properties of Tumor Hybrid Cells.Cells.2021;10(6). 19. Walker BS,Sutton TL,Zarour L,Hunter JG,Wood SG,Tsikitis VL,et al.Circulating Hybrid Cells: A Novel Liquid Biomarker of Treatment Response in Gastrointestinal Cancers.Ann Surg Oncol.2021;28(13):8567-78. 20. Lajoie MJ,Boyken SE,Salter AI,Bruffey J,Rajan A,Langan RA,et al.Designed protein logic to target cells with precise combinations of surface antigens.Science.2020;369(6511):1637-43. 21. Kloss CC,Condomines M,Cartellieri M,Bachmann M,Sadelain M.Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells.Nat Biotechnol.2013;31(1):71-5.

Claims

1. 1. A bispecific molecule comprising: (a) a first antigen-binding region that binds to a first antigen on a tumor first cell (TFC), wherein the first antigen is an epithelial cell lineage marker; and (b) a second antigen-binding region that binds to a second antigen on a TFC of the cancer, wherein the second antigen is a macrophage cell lineage marker; 2. The bispecific molecule comprising:

2. 2. The bispecific molecule of claim 1, wherein the epithelial cell lineage marker is epithelial cell adhesion molecule (EpCAM).

3. The bispecific molecule of claim 2 , wherein EpCAM comprises SEQ ID NO: 7 or SEQ ID NO:

12.

4. The bispecific molecule of claim 1 , wherein the macrophage cell lineage marker is CD163.

5. The bispecific molecule of claim 4, wherein CD163 comprises SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:

13.

6. A bispecific molecule according to any one of claims 1 to 5, wherein the first antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, the VL region comprising a light chain CDR1 (CDRL1) of SEQ ID NO: 36, a light chain CDR2 (CDRL2) of SEQ ID NO: 37, and a light chain CDR3 (CDRL3) of SEQ ID NO: 38, and the VH region comprising a heavy chain CDR1 of SEQ ID NO: 33, a heavy chain CDR2 of SEQ ID NO: 34, and a heavy chain CDR3 of SEQ ID NO:

35. Claim 7: The second antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 42, a light chain CDR2 (CDRL2) of SEQ ID NO: 43, and a light chain CDR3 (CDRL3) of SEQ ID NO: 44, and the VH region comprises a heavy chain CDR1 of SEQ ID NO: 39, a heavy chain CDR2 of SEQ ID NO: 40, and a heavy chain CDR3 of SEQ ID NO: 41; or 6. The bispecific molecule of any one of claims 1 to 5, wherein the second antigen-binding region comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region comprises a light chain CDR1 (CDRL1) of SEQ ID NO: 48, a light chain CDR2 (CDRL2) of SEQ ID NO: 49, and a light chain CDR3 (CDRL3) of SEQ ID NO: 50, and wherein the VH region comprises a heavy chain CDR1 of SEQ ID NO: 45, a heavy chain CDR2 of SEQ ID NO: 46, and a heavy chain CDR3 of SEQ ID NO:

47.

8. The bispecific molecule of claim 1, wherein the first antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 28 and a heavy chain variable (VH) region of SEQ ID NO:

27.

9. The bispecific molecule of claim 1, wherein the second antigen-binding region comprises a light chain variable (VL) region of SEQ ID NO: 30 and a heavy chain variable (VH) region of SEQ ID NO: 29, or a light chain variable (VL) region of SEQ ID NO: 32 and a heavy chain variable (VH) region of SEQ ID NO:

31.

10. The bispecific molecule of any one of claims 1 to 5, wherein the bispecific molecule is a bispecific antibody or an antigen-binding fragment thereof.

11. The bispecific molecule of claim 10 , wherein the bispecific antibody is conjugated to a drug.

12. A pharmaceutical composition comprising the bispecific molecule of any one of claims 1 to 5.

13. 10. An agent comprising the bispecific molecule of any one of claims 1 to 5 for use in treating or preventing cancer in a subject in need thereof.

14. The agent according to claim 13, wherein the cancer is a liquid cancer.

15. The method of claim 13 , wherein the cancer comprises a solid tumor.

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