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WO2024036232A2 - Bispecific antibodies and uses thereof - Google Patents

Bispecific antibodies and uses thereof Download PDF

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Publication number
WO2024036232A2
WO2024036232A2 PCT/US2023/071960 US2023071960W WO2024036232A2 WO 2024036232 A2 WO2024036232 A2 WO 2024036232A2 US 2023071960 W US2023071960 W US 2023071960W WO 2024036232 A2 WO2024036232 A2 WO 2024036232A2
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binding
kras
pigr
alk
domain
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WO2024036232A3 (en
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Jose CONEJO-GARCIA
Subir Biswas
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H Lee Moffitt Cancer Center and Research Institute Inc
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H Lee Moffitt Cancer Center and Research Institute Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the ⁇ -PIGR binding domain facilitates translocation of the antibody from the cell membrane into the cytosol. Binding of the antibody to PIGR on the cell surface causes the antibody to be trafficked in endosomes through a process termed transcytosis, until it reaches the cell membrane on a different pole, where a fragment of PIGR is cleaved. This results in a complex consisting of the bi-specific antibody, a fragment of PIGR (now termed the secretory component), and any antigen they encountered through transcytosis. Therefore, disclosed herein is a bispecific antibody having an oncogene or gene fusion-binding domain and an ⁇ -PIGR binding domain.
  • the ⁇ -PIGR binding domain comprises an ⁇ -PIGR-binding variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and an ⁇ -PIGR-binding variable light (VL) domain having CDR1, CDR2 and CDR3 sequences.
  • VH variable heavy
  • VL variable light
  • the CDR1 sequence of the ⁇ -PIGR-binding V H domain comprises the amino acid sequence GYTFIEYT (SEQ ID NO:1); the CDR2 sequence of the ⁇ -PIGR-binding V H domain comprises the amino acid sequence INPNNGGT (SEQ ID NO:2); the CDR3 sequence of the ⁇ -PIGR-binding VH domain comprises the amino acid sequence ARYYRYDVLSAMDY (SEQ ID NO:3); the CDR1 sequence of the ⁇ -PIGR-binding VL comprises the amino acid sequence ESVDNYAISF (SEQ ID NO:4); the CDR2 sequence of the ⁇ -PIGR-binding V L domain comprises the amino acid sequence AAS; and the CDR3 sequence of the ⁇ -PIGR-binding V L domain comprises the amino acid sequence QQSKAVPYT (SEQ ID NO:5).
  • the ⁇ -PIGR-binding VH domain comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFIEYTMHWVRQAPGQGLEWMGWINPN NGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARYYRYDVLSAMD YWGQGTLVTVSS (SEQ ID NO:6), and in some embodiments, the ⁇ -PIGR-binding V L domain comprises the amino acid sequence EIVLTQSPATLSLSPGERATLSCRASESVDNYAISFLAWYQQKPGQAPRLLIYAASN RATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQSKAVPYTFGQGTKLEIK (SEQ ID NO:7).
  • the oncogene is ⁇ -KRAS G12D .
  • V H oncogene-binding variable heavy
  • V L oncogene-binding variable light
  • the CDR1 sequence of the oncogene-binding VH domain comprises the amino acid sequence GTFSSYA (SEQ ID NO:8); the CDR2 sequence of the oncogene-binding VH domain comprises the amino acid sequence ISRSGHST (SEQ ID NO:9); the CDR3 sequence of the oncogene-binding V H domain comprises the amino acid sequence AKRFGSIVFDY (SEQ ID NO:10); the CDR1 sequence of the oncogene-binding V L comprises the amino acid sequence QSLFNSRTRKNY (SEQ ID NO:11); the CDR2 sequence of the oncogene-binding VL domain comprises the amino acid sequence WAS; and the CDR3 sequence of the oncogene-binding VL domain comprises the amino acid sequence KQSYYHMYT (SEQ ID NO:12).
  • the ⁇ oncogene-binding V H Attorney Docket Number: 10110-425WO1 domain comprises the amino acid sequence MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGTFSSYAMSWV RQAPGKGLEWVSTISRSGHSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCAKRFGSIVFDYWGQGTLVTVSS (SEQ ID NO:13), and in some embodiments the oncogene-binding V L domain comprises the amino acid sequence MGWSCIILFLVATATGVHSDLVMTQSPATLSLSPGERATLSCKSSQSLFNSRTRKNY LAWYQQKPGQAPRLLIYWASTRESGIPGRFSGSGSGTDFLTISSLEPEDFAVYYCK QSYYHMYTFGQGTKVEIK (SEQ ID NO:14).
  • the bi-specific antibody has the following formula: V L O – V H O – V L P – V H P, V L P – V H P – V L O – V H O, V H P – V L P – V H O – V L O, VHO – VLO – VHP – VLP, VLO – VHO – VHP – VLP, or VLP – VHP – VHO – VLO, wherein “V H O” is an oncogene or gene fusion-binding V H domain; wherein “V L O” is an oncogene or gene fusion-binding V L domain; wherein “VHP” is a ⁇ -PIGR-binding VH domain; wherein “VLP” is a ⁇ -PIGR-binding VL domain; and wherein “–” consists of a peptide linker or a peptide bond.
  • the oncogene is selected from the group consisting of ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM, BCLAF1, BRAF, BRCA1, BRCA2, CCND3, CTNNB1, CREBBP, DNMT3A, EGFR, EP300, ERBB2, ERBB3, ESR1, EZH2, FAT1, FAT3, FAT4, FBXO11, FBXW7, FGFR1, FLT3, FOXA1, GNA11, GNAQ, GNAS, GTF2I, H3F3A, HER2/NEU, HRAS, IDH1, IDH2, JAK2, KCNJ4, KDM6A, KIT, KMT2C, KMT2D, KRAS, LRP1B, MET, MTOR, MYC, MYCN, NF1, NOTCH1, NRAS, NSD1, NSD2, NTRK3, OBSCN, PCBP1, PIK3CA, PIK
  • the gene fusion is selected from the group consisting of EML4-ALK, KIF5B-ALK, HIP1-ALK, KLC1-ALK, DCTN1-ALK, PRKAR1A-ALK, STRN-ALK, CLTC-ALK, MPRIP-ALK, NPM1-ALK, TNS1-ALK, ACTG2-ALK, IGFBP5- ALK, SEC31A-ALK, TPM3-ALK, AITC-ALK, TPM4-ALK, CD74 ROS1, SLC34A2- ROS1, SDC4-ROS1, EZR-ROS1, LR1G3-ROS1, SDC4-ROS1, TPM3-ROS1, LIMA1- ROS1, MSN-ROS1, WNK1-ROS1, RBPMS-ROS1, KIF5B-RET, CCDC6-RET, and TRIM33-RET.
  • Also disclosed is a method of treating a cancer in a subject comprising administering to the subject a bispecific antibody disclosed herein. Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis associated with expression of an oncogene (such as, for example, ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM, BCLAF1, BRAF, BRCA1, BRCA2, CCND3, CTNNB1, CREBBP, DNMT3A, EGFR, EP300, ERBB2, ERBB3, ESR1, EZH2, FAT1, FAT3, FAT4, FBXO11, FBXW7, FGFR1, FLT3, FOXA1, GNA11, GNAQ, GNAS, GTF2I, H3F3A, HER2/NEU, HRAS, IDH1, IDH2, JAK2, KCNJ4, KDM6A, KIT, KMT2C
  • the dimeric antigen molecule can be administered in one or more viral vectors and self-assemble inside the cancerous cell.
  • an oncogene such as, for example, 10 ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM, BCLAF1, BRAF, BRCA1, BRCA2, CCND3, CTNNB1, CREBBP, DNMT3A, EGFR, EP300, ERBB2, ERBB3, ESR1, EZH2, FAT1, FAT3, FAT4, FBXO11, FBXW7, FGFR1, FLT3, FOXA1, GNA11, GNAQ, GNAS, GTF2I, H3F3A, HER2/NEU, HRAS, IDH1, IDH2, JAK2, KCNJ4, KDM6A, KIT, KMT
  • an oncogene such as, for example, 10 ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM
  • a dimeric antigen binding molecule comprising transfecting a cell with 3 viral vectors (such as, for example, pBMN, adeno-associated virus (AAV), Adenovirus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus (HIV), Sindbis virus, and Murine Maloney Leukemia virus) each comprising different antibody constructs; wherein a first viral construct encodes an antibody J chain; a second viral construct encodes an antibody VH chain, and a third viral construct encodes an antibody VL chain; wherein expression of the VH, VL, and J chains causes formation of a dimeric antigen binding molecule.
  • 3 viral vectors such as, for example, pBMN, adeno-associated virus (AAV), Adenovirus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus (HIV), Sindbis virus, and Murine Maloney Leukemia virus
  • Figure 1 illustrates embodiments of the disclosed bispecific antibodies.
  • Figures 2A, 2B, 2C, 2D, 2E, 2F, and 2G show KRAS G12D mutation-specific dIgA1, but not IgG4, neutralizes KRAS G12D inside tumor cells and expels the oncodriver outside tumor cells.
  • Figure 2B shows confocal immunofluorescence microscopy images showing that Alexa Fluor 647-conjugated anti-KRAS G12D -IgA1, but not with anti-KRAS G12D - IgG4, penetrates KRAS G12D -mutant A427 and SK-LU-1 NSCLC cells, while KRAS G12D -specific, but not control irrelevant dIgA, co-immunoprecipitated with Attorney Docket Number: 10110-425WO1 KRAS G12D ; Scale bar, 25 ⁇ m.
  • Figure 2C shows that OVCAR3 cells were transduced with KRAS G12D or WT KRAs fused to PAmCherry (Red).
  • FIG 2G shows Left, Transcytosis experiment adding biotinylated anti-KRAS G12D -IgA1/IgG4 or irrelevant IgA to the upper chamber of a transwell system, where physical access of antibodies to the basal chamber is prevented by WT or KRAS G12D -OVCAR3 cells grown to confluence in transwell inserts. Right, dot blots showing presence of KRAS G12D (top panel) or beads (bottom panel) in the streptavidin immunoprecipitates of the basal and upper chamber contents.
  • Figures 3A and 3B show KRAS G12D -specific dIgA1, but not the same antibody on an IgG4 backbone, captures mutant KRAS during trafficking.
  • Figure 3B shows Top panel, Non-denatured lysates (6 mg) of KRAS G12D -OVCAR3 cells treated with anti-KRAS G12D -IgA1, anti-KRAS G12D -IgG4, or vehicle (PBS) were immunoprecipitated using anti-Rab5A, Rab7A, Rab8A, or Rab11A antibodies. Immunoprecipitates were then blotted for KRAS G12D -specific IgA or IgG.
  • Figures 4A, 4B, 4C, 4D, 4E, and 4F show KRAS G12D -specific dIgA treatment reduces proliferation, without causing apoptosis, of KRAS G12D -mutated, but not KRAS WT cancer cells.
  • Figure 4A shows MTT assay-based absorbance at 570 nm of KRAS G12D -transduced OVCAR3 cells treated with irrelevant IgA or anti-KRAS G12D IgA1, relative to mean absorbance of vehicle (PBS) treated cells, at different temporal points.
  • Figures 5A, 5B, 5C, 5D, and 5E show KRAS G12D -specific dIgA1, but not the same antibody on an IgG4 backbone, abrogates KRAS G12D tumor growth in vivo.
  • Figure 5A shows a schematic of design of experiment shown in B and C. Antibody (100 ⁇ g per 20 g body weight) or equal volume of vehicle (PBS) was intratumorally (IT) injected.
  • Figure 5D shows a schematic of design of experiment.
  • Antibody 100 ⁇ g per 20 g body weight
  • PBS equal volume of vehicle
  • MRTX1133 200 ⁇ g per 20 g body weight
  • IP intraperitoneally
  • Tumor growth curves left
  • tumor weight right
  • Data are presented as mean ⁇ SEM.
  • Figure 5E shows a representative immunohistochemical staining of human IgA of KRAS G12D -OVCAR3 mice tumors treated intraperitoneally with non-antigen specific or KRAS G12D -specific dIgA. Scale bar, 400 or 60 ⁇ m, as indicated.
  • Figures 6A, 6B, 6C, 6D, 6E, 6F, and 6G show KRAS G12D -specific dIgA1 is more effective than small molecule KRAS G12D inhibitors at abrogating the progression of KRAS G12D NSCLC.
  • Figure 6A shows the combined staining of pIgR, IgA, IgG, CD3, Pan cytokeratin (panCK) and DAPI, representative of 30 clinical cases of squamous cell carcinoma or adenocarcinoma of the lung. Scale bar, 200 ⁇ m (upper panel), 100 ⁇ m (magnified lower panels).
  • Figure 6B shows the accumulation of CD3 + T cells in human panCK + NSCLC islets are associated with increased coating of tumor cells with IgA and IgG, expression of pIgR and co-localization of IgA with pIgR. Data are mean ⁇ SEM. **P ⁇ 0.01; ***P ⁇ 0.001, unpaired two-tailed t-test.
  • Figure 6C shows a schematic of design of experiment shown in D&E.
  • Antibody 100 ⁇ g per 20 g body weight
  • MRTX1133 200 ⁇ g per 20 g body weight
  • All treatments were IP injected.
  • Figure 6D shows tumor growth curves from KRAS G12D A427 tumor-bearing NSG mice receiving (IP) anti-KRAS G12D -IgA1, anti-KRAS G12D -IgG4 or vehicle every 4 days.
  • MRTX1133 was administered in different mice every 4 days (left) or daily (middle), as indicated. Tumor weight is shown on the right.
  • Figure 6E shows Western blot showing phosphorylated and total ERK1/2 in lysates of KRAS G12D -mutated A427 cells, treated with anti-KRAS G12D -IgA1 or MRTX1133.
  • Figure 6G shows Left, schematic design of treatments in Attorney Docket Number: 10110-425WO1 KPMSH2 KIN lung tumor-bearing immunocompetent mice.
  • Antibody 100 ⁇ g per 20 g body weight or equal volume of vehicle (PBS) was IP injected. When indicated, CD8 T cells were depleted with intraperitoneal anti-CD8 antibodies.
  • Tumor growth curves from mice receiving intraperitoneal vehicle or anti-KRAS G12D -dIgA1, or anti- IDH1 R132H -dIgA1, with or without CD8 T cell depletion. Growth curves were pooled from 2 independent experiments (n 8-10 mice per group). Data are mean ⁇ SEM.
  • Figures 7A, 7B, 7C, 7D, 7E, 7F, and 7G show IDH1 R132H -specific dIgA1 abrogates IDH1-mutated colon cancer growth in vivo in a PIGR expression- dependent manner.
  • Figure 7Bs shows a schematic of design of experiment shown in C-F. Antibody (100 ⁇ g per 20 g body weight) or equal volume of vehicle (PBS) was injected IP.
  • PBS vehicle
  • Figure 7D shows tumor growth curves (left), tumor weight (middle) and tumor volume of one representative experiment (right) from identically treated untransduced (PIGR low ) WT- IDH1 (IDH1 +/+ ) tumor-bearing NSG mice.
  • Figure 7F shows tumor growth curves (left), tumor weight (middle), tumor volume of one representative experiment (right) from PIGR-transduced WT-IDH1 (IDH1 +/+ ) tumor- bearing NSG mice treated as in 7C-7E.
  • FIG. 7G shows a schematic of design of experiment.
  • Monomeric or dimeric anti-IDH1 G12D - Attorney Docket Number: 10110-425WO1 IgA1, anti-IDH1 G12D -IgG4 antibody or equal volume of vehicle (PBS) were injected IP into mutant-IDH1 (IDH1 +/R132H ) tumor-bearing NSG mice. All antibodies were administered at100 ⁇ g per 20 g body weight.
  • Figures 8A, 8B, and 8C show PIGR mRNA expression across cancer types and confirmation of dimeric fraction in native IgA antibodies.
  • Figure 8A shows TCGA data showing PIGR mRNA expression, expressed as RNA Seq V2 RSEM (log2(value+1)) in several epithelial and non-epithelial malignancies.
  • Figure 8B shows native human IgA was subjected to mass spectrometry.
  • FIG. 8C shows native gel electrophoresis of two different lot of native human IgA procured from Abcam (ab91025) which shows the presence of both dimeric and monomeric IgA in the gel at their corresponding molecular weights.
  • Figures 9A, 9B, 9C, 9D, 9E, and 9F show production and purification of KRAS G12D and IDH1 R132H mutation specific recombinant dIgA1 and IgG4 antibodies.
  • Figure 9B shows graphs showing peaks of purified anti-human KRAS G12D and IDH1 R132H dIgA and IgG4 antibodies at 280nm.
  • Figure 9C shows native gel electrophoresis of different IgA antibodies that shows the presence of dimeric (black arrows) and monomeric IgA (red arrows) in the gel at their corresponding molecular weights.
  • Figure 9D shows immunoblots showing that recombinant anti-KRAS G12D - IgA1 and anti- KRAS G12D -IgG4 antibodies specifically recognize mutant KRAS G12D , in the lysates of KRAS G12D -PAmCherry-OVCAR3, but not KRAS WT -PAmCherry- OVCAR3 cells, as well as KRAS G12D mutated A427 and SK-LU-1 lung cancer cell lines.
  • Figure 9E shows immunoblots showing KRAS G12D levels in anti-KRAS G12D -IgA1/IgG4 or non-antigen specific irrelevant IgA-treated KRAS G12D -A427 lung cancer cell lysates.
  • Figure 9F shows transcytosis experiment adding biotinylated anti-KRAS G12D -IgA1/IgG4 or Attorney Docket Number: 10110-425WO1 irrelevant IgA to the upper chamber of a transwell system, where physical access of antibodies to the basal chamber is prevented by WT or KRAS G12D -OVCAR3 cells grown to confluence in transwell inserts.
  • Dot blots showing presence of KRAS G12D (left panel), human IgA (middle panel), or human IgG (right panel) in the streptavidin immunoprecipitates of the basal and upper chamber contents.
  • Figures 10A, 10B, and 10C show intratumoral infusion of KRAS G12D -specific dIgA1, but not the same antibody on an IgG4 backbone abrogates KRAS G12D - OVCAR3, but not KRAS WT -OVCAR3 tumor growth, superior to control IgA and MRTX1133, in Rag1-deficient mice.
  • Figure 10A schematic of design of experiment shown in ‘B’, and ‘C’.
  • Antibody 100 ⁇ g per 20 g body weight
  • PBS intratumorally
  • MRTX1133 200 ⁇ g per 20 g body weight
  • IP intraperitoneally
  • Figures 11A and 11B show intraperitoneal infusion of KRAS G12D -specific dIgA1, but not the same antibody on an IgG4 backbone abrogates KRAS G12D - OVCAR3, but not KRAS WT - OVCAR3 tumor growth, superior to control IgA and MRTX1133, in NSG mice.
  • Figure 11A shows a schematic of design of experiment shown in ‘B’.
  • Antibody 100 ⁇ g per 20 g body weight or equal volume of vehicle (PBS) every 4 d or MRTX1133 (200 ⁇ g per 20 g body weight) every 4 d or daily were injected intraperitoneally (IP).
  • Figures 12A, 12B, 12C, 12D, AND 12E show intraperitoneal infusion of KRAS G12D -specific dIgA1, but not the same antibody on an IgG4 backbone abrogates Attorney Docket Number: 10110-425WO1 KRAS G12D mutated A427 or SK-LU-1 tumors, superior to control IgA and MRTX1133, in NSG mice.
  • Figure 12B shows a schematic of design of experiment shown in ‘C’ and ‘D’.
  • Antibody 100 ⁇ g per 20 g body weight) or equal volume of vehicle (PBS) every 4 d or MRTX1133 (200 ⁇ g per 20 g body weight) daily or every 4 d were injected IP.
  • Figure 12E shows Left, schematic design of treatments in KRAS G12D Brpkp110 tumor-bearing immunocompetent mice. Antibody (100 ⁇ g per 20 g body weight) were intraperitoneally injected.
  • Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.
  • the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts Attorney Docket Number: 10110-425WO1 by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.
  • An “increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount.
  • the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.
  • a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth).
  • “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.
  • prevent or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce.
  • the term “subject” refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline.
  • the subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • therapeutically effective refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific Attorney Docket Number: 10110-425WO1 therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • Biocompatible generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.
  • “Comprising” is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others.
  • compositions and methods shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of'' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure. A “control” is an alternative subject or sample used in an experiment for comparison purposes.
  • a control can be "positive” or "negative.”
  • Effective amount of an agent refers to a sufficient amount of an agent to provide a desired effect.
  • the amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.
  • an “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a "pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated Attorney Docket Number: 10110-425WO1 into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained.
  • “Pharmaceutically acceptable carrier” means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use.
  • carrier or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
  • carrier encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.
  • “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
  • “Therapeutic agent” refers to any composition that has a beneficial biological effect.
  • Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer).
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • a therapeutic agent refers to an agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
  • Attorney Docket Number: 10110-425WO1 “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result.
  • a desired therapeutic result is the control of type I diabetes.
  • a desired therapeutic result is the control of obesity.
  • Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject.
  • the term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief.
  • the precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
  • compositions Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc.
  • Antibodies Generally in some aspects, the disclosed antigen binding molecules are antibodies.
  • the term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies.
  • antibodies In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with an oncogene such that the oncogene is bound in the cytosol of a cell and secreted outside the cell while being bound to said oncogene.
  • the antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods.
  • IgA human immunoglobulins
  • IgD immunoglobulins
  • IgE immunoglobulins
  • IgG immunoglobulins
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.
  • the disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the monoclonal antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S.
  • Digestion of antibodies to produce fragments thereof, particularly, Fab fragments can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec.22, 1994 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a Attorney Docket Number: 10110-425WO1 residual Fc fragment.
  • antibody or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab, Fv, scFv, VHH, and the like, including hybrid fragments.
  • fragments of the antibodies that retain the ability to bind their specific antigens are provided.
  • antibody or fragment thereof fragments of antibodies which maintain oncogene (such as KRAS G12D ) binding activity are included within the meaning of the term “antibody or fragment thereof.”
  • Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).
  • conjugates of antibody fragments and antigen binding proteins single chain antibodies.
  • the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non- modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.
  • the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen.
  • Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
  • antibody or “antibodies” can also refer to a human antibody and/or a humanized antibody.
  • Many non-human antibodies e.g., those Attorney Docket Number: 10110-425WO1 derived from mice, rats, or rabbits
  • 10110-425WO1 derived from mice, rats, or rabbits
  • Human antibodies Human antibodies can be prepared using any technique.
  • the disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)).
  • the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge.
  • Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein.
  • Humanized antibodies Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.
  • a humanized form of a non-human antibody is a chimeric antibody or antibody chain (or a fragment thereof, such as an sFv, Fv, Fab, Fab’, F(ab’)2, or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non- human (donor) antibody integrated into the framework of a human (recipient) antibody.
  • a humanized antibody residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen).
  • Fv framework (FR) residues of the human antibody are replaced by corresponding non- Attorney Docket Number: 10110-425WO1 human residues.
  • Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).
  • Fc antibody constant region
  • humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • Methods that can be used to produce humanized antibodies are also described in U.S. Patent No.4,816,567 (Cabilly et al.), U.S. Patent No. 5,565,332 (Hoogenboom et al.), U.S.
  • Patent No.5,721,367 (Kay et al.), U.S. Patent No.5,837,243 (Deo et al.), U.S. Patent No.5, 939,598 (Kucherlapati et al.), U.S. Patent No.6,130,364 (Jakobovits et al.), and U.S. Patent No.6,180,377 (Morgan et al.).
  • Administration of antibodies Administration of the antibodies can be done as disclosed herein. Nucleic acid approaches for antibody delivery also exist.
  • the broadly neutralizing antibodies and antibody fragments can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment.
  • a nucleic acid preparation e.g., DNA or RNA
  • the delivery of the nucleic acid can be by any means, as disclosed herein, for example.
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
  • Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math.2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol.48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci.
  • the ⁇ -PIGR-binding antibody can comprise a variable heavy (V H ) domain having CDR1, CDR2 and CDR3 sequences and a variable light (VL) domain having CDR1, CDR2 and CDR3 sequences.
  • V H variable heavy
  • VL variable light
  • the CDR1 sequence of the ⁇ -PIGR-binding VH domain comprises the amino acid sequence GYTFIEYT (SEQ ID NO:1); the CDR2 sequence of the ⁇ -PIGR-binding V H domain comprises the amino acid sequence INPNNGGT (SEQ ID NO:2); the CDR3 sequence of the ⁇ -PIGR-binding V H domain comprises the amino acid sequence ARYYRYDVLSAMDY (SEQ ID NO:3); the CDR1 sequence of the ⁇ -PIGR-binding VL comprises the amino acid sequence ESVDNYAISF (SEQ ID NO:4); the CDR2 sequence of the ⁇ -PIGR-binding VL domain comprises the amino acid sequence AAS; and the CDR3 sequence of the ⁇ -PIGR-binding VL domain comprises the amino acid sequence QQSKAVPYT (SEQ ID NO:5).
  • the ⁇ -PIGR-binding VH domain comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFIEYTMHWVRQAPGQGLEWMGWINPN NGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARYYRYDVLSAMD YWGQGTLVTVSS (SEQ ID NO:6).
  • the ⁇ -PIGR-binding VL domain comprises the amino acid sequence EIVLTQSPATLSLSPGERATLSCRASESVDNYAISFLAWYQQKPGQAPRLLIYAASN RATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQSKAVPYTFGQGTKLEIK (SEQ ID NO:7).
  • the heavy and light chains are preferably separated by a linker. Suitable linkers for scFv antibodies are known in the art.
  • the linker comprises the amino acid sequence GGGGS (SEQ ID NO:15), GGGGSGGGGS (SEQ ID NO:16) or GGGGSGGGGSGGGGS (SEQ ID NO:17).
  • the scFv can have the formula NH3-VH-linker-VL-COOH or NH3-VL-linker-VH-COOH.
  • Anti-oncogene antibody In some embodiments, the oncogene is ⁇ -KRAS G12D .
  • the oncogene-binding domain comprises a oncogene-binding variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a oncogene-binding variable light (VL) domain having CDR1, CDR2 and CDR3 sequences.
  • the CDR1 sequence of the oncogene-binding VH domain comprises the amino acid sequence GTFSSYA (SEQ ID NO:8); the CDR2 sequence of the oncogene- binding V H domain comprises the amino acid sequence ISRSGHST (SEQ ID NO:9); the CDR3 sequence of the oncogene-binding V H domain comprises the amino acid sequence AKRFGSIVFDY (SEQ ID NO:10); the CDR1 sequence of the oncogene- binding VL comprises the amino acid sequence QSLFNSRTRKNY (SEQ ID NO:11); the CDR2 sequence of the oncogene-binding VL domain comprises the amino acid sequence WAS; and the CDR3 sequence of the oncogene-binding V L domain comprises the amino acid sequence KQSYYHMYT (SEQ ID NO:12).
  • the oncogene-binding V H domain comprises the amino acid sequence MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGTFSSYAMSWV RQAPGKGLEWVSTISRSGHSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCAKRFGSIVFDYWGQGTLVTVSS (SEQ ID NO:13).
  • the oncogene-binding VL domain comprises the amino acid sequence MGWSCIILFLVATATGVHSDLVMTQSPATLSLSPGERATLSCKSSQSLFNSRTRKNY LAWYQQKPGQAPRLLIYWASTRESGIPGRFSGSGSGTDFLTISSLEPEDFAVYYCK QSYYHMYTFGQGTKVEIK (SEQ ID NO:14).
  • Pharmaceutical carriers/Delivery of pharmaceutical products As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant.
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • the exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like.
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in Attorney Docket Number: 10110-425WO1 liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No.3,610,795, which is incorporated by reference herein.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
  • Pharmaceutically Acceptable Carriers The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • an appropriate amount of a pharmaceutically- acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and Attorney Docket Number: 10110-425WO1 injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable..
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual Attorney Docket Number: 10110-425WO1 physician in the event of any counterindications.
  • Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch.22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp.365- 389.
  • a typical daily dosage of the antibody used alone might range from about 1 ⁇ g/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • Methods of using the compositions The disclosed compositions can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers.
  • Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis associated with expression of an oncogene such as, for example, ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM, BCLAF1, BRAF, BRCA1, BRCA2, CCND3, CTNNB1, CREBBP, DNMT3A, EGFR, EP300, ERBB2, ERBB3, ESR1, EZH2, FAT1, FAT3, FAT4, FBXO11, FBXW7, FGFR1, FLT3, FOXA1, GNA11, GNAQ, GNAS, GTF2I, H3F3A, HER2/NEU, HRAS, IDH1, IDH2, JAK2, KCNJ4, KDM6A, KIT, KMT2C, KMT2D, KRAS, LRP1B, MET, MTOR, MYC, MYCN, NF1,
  • the dimeric antigen molecule can be administered in one or more viral vectors and self-assemble inside the cancerous cell.
  • an oncogene such as, for example, ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM, BCLAF1, BRAF, BRCA1, BRCA2, CCND3, CTNNB1, CREBBP, DNMT3A, EGFR, EP300, ERBB2, ERBB3, ESR1, EZH2, FAT1, FAT3, FAT4, FBXO11, FBXW7, FGFR1, FLT3, FOXA1, GNA11, GNAQ, GNAS, GTF2I, H3F3A, HER2/NEU, HRAS, IDH1, IDH2, JAK2, KCNJ4, KDM6A, KIT, KMT2
  • an oncogene such as, for example, ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM,
  • a representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder Attorney Docket Number: 10110-425WO1 cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers such as small cell lung cancer and non- small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon cancer, rectal cancer, prostatic cancer, or pancreatic cancer
  • the disclosed treatments methods can also include the administration any anti- cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin- stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado- Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride),
  • Treatment methods can include or further include checkpoint inhibitors include, but are not limited to antibodies that block PD-1 (Pembrolizumab, Nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (Ipilimumab (MDX- 010), Tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7- H4, TIM3, LAG-3 (BMS-986016).
  • PD-1 Pieriszumab
  • Nivolumab BMS-936558 or MDX1106
  • CT-011, MK-3475 PD-L1
  • PDX-1105 BMS-936559
  • MPDL3280A MPDL3280A
  • MSB0010718C MSB0010718C
  • Dimeric IgA specifically disables intracellular mutated oncodrivers Limited by their large size and considered unable to the development of antibody-based immunotherapies has been transmembrane or extracellular targets.
  • Sotorasib a specific KRAS G12C small molecule inhibitor, has been recently recently approved by the FDA.
  • inhibitors against KRAS G12D the more common mutation in pancreatic and colon cancer, are currently under testing.
  • IgA transcytoses through endosomes, which could prevent direct contact with specific antigens inside tumor cells. Even if transcytosing IgA is not “shielded” by endosomes, targeted antigens could not co-localize inside tumor cells. Experimentation with these processes can determine if it is possible for engineered Attorney Docket Number: 10110-425WO1 dIgA to target intracellular molecules, with important therapeutic implications: First, the mechanisms of resistance that invariably follow the administration of small molecule inhibitors could be still sensitive to antibody-mediated neutralization, and vice versa. Second, the half-life of unmodified IgA ⁇ 6 days, but can be further optimized to increase stability.
  • dIgA is particularly abundant at mucosal surfaces, suggesting that exogenous dIgA could be safely administered.
  • Our results provide a rationale for developing dIgA-based therapeutics to neutralize various intracellular antigens in human cancer and other diseases.
  • RESULTS Mutation-specific dIgA neutralizes intracellular KRAS G12D and expels it outside tumor cells, without effects on KRAS WT cells.
  • PIGR-mediated dIgA transcytosis we first analyzed PIGR expression in tumors in TCGA datasets.
  • KRAS G12D -mutated lung cancer cells Fig.2B, left
  • mutation-specific, but not control dIgA targeted KRAS
  • Fig.2B right
  • KRAS G12D -specific dIgA penetrated OVCAR3 cells transduced with KRAS WT or KRAS G12D fused to photoactivatable (PA)m-Cherry fluorescent protein, but only disrupts cytoplasmic localization and distribution of intracellular KRAS G12D (Fig.2C).
  • PA photoactivatable
  • mutant KRAS was not only neutralized inside tumor cells, but also expelled outside the cell through transcytosis, as evidenced by co-occurrence of KRAS G12D with secretory IgA in the supernatant of KRAS G12D -PAmCherry-transduced OVCAR3 cells upon treatment with KRAS G12D -specific dIgA (Fig.1D), with decreased intracellular KRAS G12D (Fig.2E & Fig9E).
  • PAmCherry fragments, fused to KRAS G12D were only found in the supernatant of tumor cells treated with KRAS G12D -specific dIgA in 3 independent experiments (Fig.2F, left).
  • KRAS G12D was not found in supernatants upon treatment with KRAS G12D -specific IgG4, irrelevant IgA containing dimers, or vehicle (Fig.2D&2F, left).
  • PIGR was identified in the supernatants of cells treated with antigen-specific or control dIgA (Fig.2F, right).
  • tumor cell- penetrating dIgA can indeed target specific mutations in KRAS inside tumor cells, resulting in intracellular decreased levels and expelling the oncodriver outside the tumor cell, without obvious effects in KRAS unmutated epithelial cells.
  • KRAS G12D and mutation-specific dIgA are found in the same endosomal compartments upon transcytosis.
  • mutant KRAS was primarily found in RAB8A + /RAB11A + recycling endosomes when tumor cells were treated with vehicle or antigen-specific IgG (Fig.3B).
  • treatment with KRAS G12D -specific dIgA concentrated mutant KRAS on the same endosomes through which dIgA traffics inside tumor cells (Fig.3B) indicate that transcytosing dIgA encounters the antigen on the cell membrane and hauls it in endosomes, eventually expelling the target through secretory IgA.
  • dIgA traffics through endosomes, dIgA retains its capacity to specifically target intracellular antigens; at least those that are located near the cell membrane. Consistent with capture and extracellular disposal of KRAS G12D , treatment of KRAS G12D -transduced cancer cells with mutation-specific dIgA inhibited tumor cell proliferation, without effects on the same cells transduced with KRAS WT or untransduced ovarian cancer cells (Fig.4A-C).
  • KRAS G12D -specific dIgA specifically abrogates the progression of mutant tumors in vivo
  • Irrelevant dIgA dampens the RAS pathway by upregulating DUSP phosphatases that dephosphorylate ERK1/2, while activating inflammatory pathways associated with IFN and TNF signaling, as well as pro- apoptotic pathways linked to ER stress. Accordingly, non-specific dIgA delayed the growth of established ovarian tumors.
  • Fig.6C&6D 5 intraperitoneal injections of KRAS G12D -specific dIgA, or 20 daily treatments of MRTX1133 at high dose (200 ⁇ g), effectively abrogated the Attorney Docket Number: 10110-425WO1 progression of established A427 NSCLC, while the same antibody on an IgG4 backbone had negligible effects (Fig.6C&6D; Fig.12B&12C). Accordingly, treatment with both KRAS G12D -specific dIgA and MRTX1133 decreased ERK1/2 phosphorylation in A427 cells in a time-dependent manner (Fig.6E).
  • KRAS G12D - mutant KPMSH2 KIN lung tumors generated by cloning lung cancer cells from C57/BL6 mice with inducible mutations, activated using intra-nasal adenoviral Cre; plus restoring MSH2 through lentiviral transduction.
  • dIgA targeting KRAS G12D also elicited a significant delay in tumor growth (Fig.6G).
  • anti-tumor effects in this immunogenic system were dependent on CD8 T cells (Fig.6G), highlighting the complexity of a coordinated adaptive immune response.
  • dIgA is effective against mutations located deeper into the cytoplasm of tumor cells, in a PIGR-dependent manner
  • dIgA1 or IgG4 specifically targeting the cytoplasmic R132H mutation in isocitrate dehydrogenase 1 (IDH1), a common hotspot in multiple human tumors (Fig.7A; Fig.9A-9C).
  • IDH1 WT and IDH1 +/R132H HCT116 colon cancer cells which express low PIGR levels, and transduced them with PIGR, resulting in expression levels comparable to those found in multiple carcinoma cell lines (Fig.7A).
  • Low PIGR Attorney Docket Number: 10110-425WO1 expression abrogated the non-specific anti-tumor effects of irrelevant IgA in this system (Fig.7B-7D).
  • IDH1 R132H -specific dIgA delayed tumor growth in mutant tumors, without effect in the absence of IDH1 R132H mutations (Fig.7C&7D).
  • anti-tumor effects were dependent on PIGR expression in tumor cells, because the administration of IDH1 R132H -specific dIgA resulted in a 3.7- fold reduction in tumor growth, compared to vehicle, when PIGR-transduced IDH1 R132H HCT116 cells were treated, compared to a 1.8-fold reduction in low-PIGR- expressing parental IDH1 R132H HCT116 colon tumors (Fig.7B, 7C, & 7E).
  • anti-tumor effects were again mutation-specific, because a comparable reduction in tumor growth was observed when mice with IDH1 WT tumors were treated with irrelevant IgA or IDH1 R132H -specifc dIgA (Fig.7F).
  • PIGR-mediated transcytosis of dIgA also occurs in non-gynecological carcinomas, including lung and colorectal cancers.
  • PIGR is expressed in most epithelial malignancies, but only at lower levels in non- epithelial cancers, this mechanism can be relevant for most human epithelial cancers.
  • PIGR-mediated dIgA transcytosis progresses through endosomal trafficking but their route of trafficking may slightly differ in malignant cells than in non-malignant cells 8 . Despite trafficking through endosomes, dIgA retains its capacity to specifically target intracellular antigens.
  • Novel studies can determine whether other commonly mutated oncogenes such as PI3K or AKT, or immunosuppressive intracellular pathways such as IDO, can be more effectively targeted with dIgA than with small molecule inhibitors.
  • dIgA-mediated targeting, along or in combination with small molecules can have significant advantages, compared to small molecules: First, our results underscore the specificity of this approach. Second, although the half-life of IgA ( ⁇ 6 days in primates and >15 hrs in mice) is lower than that of IgG, and there can be significant differences in availability of PIGR-binding dimeric IgA vs.
  • dIgA expels the mutated oncogene outside tumor cells, which is obviously advantageous, compared to temporary neutralization.
  • the prospect of oral secretory IgA treatment can make this intervention ideal against tumors of the digestive tract. We therefore propose a rationale for using dIgA as a new form of immunotherapy to target intracellular oncodrivers, which opens multiple new avenues to treat otherwise undruggable carcinomas.
  • mice bearing syngeneic immunogenic carcinomas further supports the concept that the interaction between human dIgA and mouse pIgR mirrors the bioavailability of antigen-specific dIgA in vivo, in the presence of PIGR + healthy epithelial cells that can compete for the antibody.
  • mice do not express CD89, which also binds IgA and could be partially responsible for enlarged persistence of IgA in primates, compared to mice, and therefore retain dIgA in circulation for much longer.
  • IgA-IgG treatment has already been safely used to treat children with chronic non-specific diarrhea, and polyclonal antibody preparations containing >20% IgA can be safely administered in patients with severe pneumonia.
  • mutation-specific dIgA had no effect on wild-type cells, while dimeric IgA is the predominant Ig at mucosal secretions. It is therefore unlikely that IgA administration can cause on-target, off-tumor toxicity.
  • FDA-approved KRAS G12C small molecule inhibitors already elicit grade 3-4 toxicity in >11% of patients.
  • OVCAR3 (RRID:CVCL_0465), A427 (RRID:CVCL_1055), SK-LU-1 (RRID:CVCL_0629), HEK293T (RRID:CVCL_0063), NCI-H23 (RRID:CVCL_1547), NCI-H647 (RRID:CVCL_1574) cell lines were purchased from ATCC (Manassas, VA). IDH1 +/+ and IDH1 R132H/+ HCT116 (RRID:CVCL_0291) cell lines were purchased from Horizon discovery. PIGR-ablated OVCAR3 cells was generated in the lab.
  • Brpkp110 was established in the lab and KPMSH2 KIN is a new model of lung cancer developed also in our laboratories that carries the same epitope recognized in human G12D-mutated KRAS.
  • Cell lines were routinely cultured in R10 (RPMI-1640, 10% FBS, penicillin (100 IU ml ⁇ 1 ), streptomycin (100 ⁇ g ml ⁇ 1 ), L-glutamine (2 mM), sodium pyruvate (0.5 mM)) media (Thermo).
  • HEK293T cell line was routinely cultured in D10 Attorney Docket Number: 10110-425WO1 (DMEM, 10% FBS, penicillin (100 IU ml ⁇ 1 ), streptomycin (100 ⁇ g ml ⁇ 1 ), L-glutamine (2 mM), sodium pyruvate (0.5 mM)) media (Thermo).Cell lines were routinely tested for negative mycoplasma contamination. Animal models For mouse models, three different strains were used.
  • mice Female, 4-6 weeks old Rag1-deficient (Rag1 -/- ) mice (RRID:IMSR_JAX:003729) and female, 7 weeks old C57BL/6J mice (RRID:IMSR_JAX:000664) were purchased from The Jackson Laboratory. NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice (RRID:IMSR_JAX:005557), originally procured from The Jackson Laboratory, were maintained by the Moffitt Cancer Center animal facility.
  • Virus production and transduction Lentiviral particles were generated by co-transfecting HEK 293T/17 (ATCC) with pLVX-IRES-PIGR-ZsGreen, or pLenti-TO/CMV-PAmCherry1-KRAS (G12D mutated KRAS or WT) and packaging/envelope vector pMD2.G (RRID:Addgene_12259) and psPAX2 (RRID:Addgene_12260) using Lipofectamine- 3000 (Invitrogen) and the viral supernatant was harvested 48h after.5 ⁇ 10 5 HCT116 or OVCAR3 cells were seeded in a 6-well plate with 3mL of the corresponding 0.45 ⁇ filtered viral supernatant with added 10 ⁇ g/mL polybrene (Millipore).
  • Tumor Models Flank tumors were initiated by injecting 1 ⁇ 10 7 KRAS WT or G12D transduced OVCAR3 or A427; or 5 ⁇ 10 6 IDH1 WT or Mut HCT116 cells or 5 ⁇ 10 5 Brpkp110 cells; or 1 ⁇ 10 6 KPMSH2 KIN cells in PBS.
  • SK-LU-1 tumors were initiated by injecting 1 ⁇ 10 7 cells in PBS admixed with 1:1 Matrigel into flanks. Tumor volume was calculated as: 0.5 ⁇ (L ⁇ W 2 ), where L is length and W is width.
  • Intratumoral or peritumoral injections, as well as intraperitoneal injections of antibodies were done on multiple times at 3-4 days interval starting from day 7 for OVACR3, A427, HCT116, Brpkp110 and KPMSH2 KIN tumors or from day 20 for SK- LU-1 tumors after the tumor challenge at a dose of 100 ⁇ g/20g.
  • MRTX1133 (MedChemExpress, #HY-134813) was injected daily or multiple times at 3-4 days interval starting from day 7 for OVACR3 and A427 or from day 20 for SK-LU-1 tumors at a dose of 200 ⁇ g/20g.
  • CD8 T cell depletion has been performed using ⁇ CD8 antibodies (BioXCell), injected at same frequency of injected antibodies, at a dose of 200 ⁇ g/20g.
  • Recombinant antibody production Anti-KRAS G12D , and anti-IDH1 R132H antibody variable heavy (VH) and variable light (VL) sequences were obtained from the patent US10844136B2 and CN106957367A, respectively.
  • VH sequences followed by human IgA1 or IgG4 heavy chain constant region sequences in frame were cloned into PBMN-I-GFP vector (RRID:Addgene_1736).
  • VL sequences along with human kappa light chain constant region sequences in frame were cloned into pVITRO1 (Genscript).
  • Vectors, coding for all the VH and VL, were produced by Genscript. J-chain encoding pcDNA3.0 vector was procured from Addgene (RRID:Addgene_145146).
  • HEK293T cells were retrovirally transduced with antibody heavy chain encoding PBMN-I-GFP vector and sorted for viable GFP + cells, followed by transfection with antibody light chain encoding pVitro1 vector and selected using Hygromycin, and finally those cells were transfected with J-chain encoding pcDNA3.0 vector and selected using G418.
  • Heavy chain plus light chain plus J-chain expressing HEK293T cells were grown in suspension for 5-7 days in Free-Style 293 expression Attorney Docket Number: 10110-425WO1 media (Thermo) for dimeric KRAS G12D -IgA antibody production.
  • HEK293T cells expressing only light chain and respective heavy chain were used.
  • Supernatants were concentrated through a 100 kDa membrane, to eliminate any possible contaminants of unassembled J-chains, heavy chains or light chains, and antibodies were purified using IgA or IgG purification columns (Ligatrap).
  • IgA antibodies Dimerization of IgA antibodies were confirmed by detecting J-chain in the elutes through mass spectrometry. All transfections were performed using Lipofectamine 3000 (Thermo). Cell lines were routinely tested for negative mycoplasma contamination. Dimeric and monomeric IDH1 R132H -IgA1, and monomeric IDH1 R132H -IgG4 antibodies and some additional quantity KRAS G12D -IgG4 antibodies were procured from GenScript. Fluorescence-activated cell sorting (FACS) Sorting of antibody heavy chain-transduced HEK293T cells (GFP expressing) were performed by staining with DAPI (Thermo Scientific) viability dye and gated for DAPI-GFP + cells.
  • FACS Fluorescence-activated cell sorting
  • PIGR-transduced HCT116 cells were sorted by gating DAPI-GFP + cells. Samples were subsequently fluorescence-activated cell sorting (FACS) sorted using BD FACS ARIA.
  • FACS fluorescence-activated cell sorting
  • One hundred thousand KRAS G12D transduced OVCAR3 cells were placed in 6-well plates. After 12 h wells were washed, and fresh medium was added, and cells were treated with anti-human KRAS G12D -IgA1 or KRAS G12D -IgG4 or non-specific human IgA (Abcam, ab91025) at 0.5 ⁇ g/ml final concentration or vehicle (PBS) and incubated for 16 h in a 37-°C incubator.
  • Total apoptotic cells (annexin V + propidium iodide +/ ⁇ ) were analyzed by flow cytometry.
  • MTT assay Five thousand OVCAR3 (wild-type or G12D mutated KRAS-transduced or untraduced) or SK-LU-1 cells, or five hundred HEK293T cells were seeded in each well of 96-well plates.
  • PAmCherry was photoactivated using the Leica TCS SP8 DAPI bandpass filter cube (Excitation BP 450-490 nm) using the widest aperture. Exposure time was dependent on the day of the experiment and varied between 20 to 30 seconds and was optimized using positive and negative controls. The same exposure time was used for all treatments. The gain for the Hoechst channel was adjusted to compensate for photobleaching due the exposure time required for photoactivating PAmCherry. Images were acquired with a 40X/1.3 NA objective in a confocal microscope (Leica SP8) using LAS X (v.3.5.5.19976) software.
  • Proteins were extracted from the conditioned medium, reduced by DTT, digested by trypsin, and subjected to mass spectrometry analysis by the Moffitt Cancer Center Proteomics Facility. MaxQuant (version 1.5.2.8) was used to analyze the data, identify, and quantify the proteins.
  • Western blot, Native gel electrophoresis and Co-immunoprecipitation (Co-IP) Cells were lysed in RIPA buffer (Thermo) with protease-phosphatase inhibitor cocktail (CST, #5872S) and cleared by centrifugation. Proteins were quantified by Attorney Docket Number: 10110-425WO1 BCA assay (Thermo).
  • Membranes were blotted with recombinantly produced anti- KRAS G12D -IgA1 or anti-KRAS G12D -IgG4 or anti-IDH1 R132H -IgA1 or anti-IDH1 R132H -IgG4 antibodies; or commercial anti-mCherry Rockland, #600-401-P16, RRID:AB_2614470), anti-human immunoglobulin heavy alpha chain (Thermo Fisher Scientific, #A18781, RRID:AB_2535558), or anti-human immunoglobulin heavy gamma (plus light) chain (Thermo Fisher Scientific, #A18805, RRID:AB_2535582), or anti-human immunoglobulin kappa chain (R&D Systems, #MAB10050), or anti- human J-chain (Thermo Fisher Scientific, Mc19-9, #MA1-80527, RRID:AB_934333), or anti-human Rab5A (Pro
  • Lysates of A427 cells treated with KRAS G12D -specific IgA or MRTX1133 for different time points were probed with antibodies against total ERK1/2 (CST, #9102, RRID:AB_330744) and phosphorylated ERK1/2 (CST, D13.14.4E, #9102, RRID:AB_330744). Blots were then probed with appropriate horse radish peroxidase (HRP)-conjugated secondary antibodies (room temperature ,1-2 hour), unless the primary antibodies are HRP-conjugated, and immunoreactive bands were developed using Enhanced Chemiluminescence (ECL) substrate (GE HealthCare).
  • HRP horse radish peroxidase
  • the gel was scanned in a Canon TR4700 series HTTP.
  • cells were lysed using non-reducing non- denaturing lysis reagent provided in the co-immunoprecipitation (co-IP) kit (Pierce, Cat#26149) used.
  • co-IP co-immunoprecipitation
  • Proteins were immunoprecipitated using anti-human PIGR- secretory component (Abcam, SC-05, #ab3924, RRID:AB_2261963) or anti-human Rab5A, or anti-human Rab7A, or anti-human Rab8A, or anti-human Rab11A, or anti- Attorney Docket Number: 10110-425WO1 human IgA (Abcam, EPR5367-76, #ab124716, RRID:AB_10976507).
  • Proteins from concentrated conditioned media from anti-human KRAS G12D -IgA1 or KRAS G12D -IgG4 or non-specific human IgA or vehicle treated OVCAR3 cells were immunoprecipitated using anti-human PIGR-secretory component. The elutes were ran for western blotting.
  • Transwell and transcytosis assay for expelling of KRAS G12D KRAS G12D OVCAR3 cells (2 ⁇ 10 5 ) were seeded in the upper chamber of a transwell system (CORNING® 3413 TRANSWELL® 6.5mm Polycarbonate, membrane Inserts Pre-Loaded in 24-Well Culture Plates, Pore Size: 0.4 ⁇ m) in 200uL.
  • CORNING® 3413 TRANSWELL® 6.5mm Polycarbonate, membrane Inserts Pre-Loaded in 24-Well Culture Plates, Pore Size: 0.4 ⁇ m 1 mL of fresh medium was added in the basal chamber.
  • the cells were cultured in the incubator at 37 o C and 5% of CO2 during 3 days to create a monolayer.
  • KRAS G12D IgA1 or KRAS G12D IgG4 or non-specific human IgA antibodies were biotinylated using a Biotinylation Kit (Fast, Type B) by following the manufacture’s protocol. Then, 1 ⁇ g of respective biotinylated antibody was added to the upper chamber. After overnight incubation, the supernatant from the upper and basal chambers were collected into separate microcentrifuge tubes and subjected to streptavidin pull-down using Dynabeads MyOne streptavidin T1. Briefly, 10 ⁇ l of beads per sample were washed according to the manufacture’s recommendation, added to each sample and then incubated overnight at 4 o C with head-over rotation.
  • the beads were collected my magnetic separation for 5 min and washed 3 times in PBS containing 0.1% BSA. Finally, the beads were resuspended in 100uL of PBS and used for performing dot blots.
  • dot blots 10 ⁇ L of beads were blotted onto a nitrocellulose membrane and dried at room temperature 1-2 hours. Then, membranes were blocked using 5% BSA in TBS-T with a gentle agitation for 1 hour at room temperature.
  • Appropriate primary antibodies ⁇ human-IgA-HRP, ⁇ human-IgG-HRP and ⁇ KRAS G12D ) were added immediately and incubated 1 hour at room temperature with gentle rotation.
  • blots were directly chemiluminescent-developed when probed with HRP conjugated antibodies ⁇ human-IgA/IgG antibodies, or first incubated with a secondary antibody and developed.
  • Multiplex immunohistochemistry Attorney Docket Number: 10110-425WO1 FFPE tissue microarray was immunostained using the PerkinElmer OPAL TM Automation IHC kit (Waltham, MA) on the BOND RX autostainer (Leica Biosystems, Vista, CA) and the following anti-human antibodies: IgA (Abcam, EPR5367-76, ab124716, RRID:AB_10976507; 1:1000), IgG (Abcam, EPR4421, ab109489, RRID:AB_10863040; 1:500), PIGR (Abcam, ab96196, RRID:AB_10677612; 1:100), CD3 (Thermo Fisher Scientific; Cat# MA5-14524, Clone SP7; RRID: AB_109820
  • TCGA Cancer Genome Atlas
  • RNA Seq V2 RSEM RNA Seq V2 RSEM
  • Firehose Legacy for several human cancer types were analyzed and graphically exported from the cBio Cancer Genomics Portal.
  • Statistical analyses Unless mentioned otherwise all data are presented as mean ⁇ SEM. Two- tailed t-tests (unpaired and paired, as appropriate) were performed between two groups, and one-way ANOVA were performed for comparisons between more than two groups, unless indicated otherwise.
  • IgA strikes twice in ovarian cancer. Nat Rev Cancer 21, 215. 10.1038/s41568-021-00342-4. Hong, D.S., Fakih, M.G., Strickler, J.H., Desai, J., Durm, G.A., Shapiro, G.I., Falchook, G.S., Price, T.J., Sacher, A., Denlinger, C.S., et al. (2020). KRAS(G12C) Inhibition with Sotorasib in Advanced Solid Tumors. N Engl J Med 383, 1207-1217. 10.1056/NEJMoa1917239. Leusen, J.H. (2015). IgA as therapeutic antibody.
  • Anti-CD20 IgA can protect mice against lymphoma development: evaluation of the direct impact of IgA and cytotoxic effector recruitment on CD20 target cells. Haematologica 97, 1686- 1694.10.3324/haematol.2011.061408. Richards, A., Baranova, D., and Mantis, N.J. (2022). The prospect of orally administered monoclonal secretory IgA (SIgA) antibodies to prevent enteric bacterial infections. Human vaccines & immunotherapeutics 18, 1964317. 10.1080/21645515.2021.1964317. Schmick, M., Kraemer, A., and Bastiaens, P.I. (2015). Ras moves to stay in place.

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Abstract

Disclosed are bi-specific antibodies that bind an oncogene-binding domain and an α-PIGR binding domain. As disclosed herein, the α-PIGR binding domain facilitates translocation of the antibody from the cell membrane into the cytosol. Binding of the antibody to PIGR on the cell surface causes the antibody to be trafficked in endosomes through a process termed transcytosis, until it reaches the cell membrane on a different pole, where a fragment of PIGR is cleaved. This results in a complex consisting of the bi-specific antibody, a fragment of PIGR (now termed the secretory component), and any antigen they encountered through transcytosis.

Description

Attorney Docket Number: 10110-425WO1 BISPECIFIC ANTIBODIES AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS This Application claims the benefit of U.S. Provisional Application No. 63/370,798, filed on August 9, 2022, and U.S. Provisional Application No. 63/396,948, filed on August 10, 2022; applications which are incorporated herein by reference in their entireties. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government Support under Grant No. CA232758 and CA266947 awarded by the National Institutes of Health. The Government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING The sequence listing submitted on August 9, 2023, as an .XML file entitled “10110_425WO1_ST26XML” created on August 9, 2023, and having a file size of 16,223 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). BACKGROUND Despite the investment of billions of dollars and years of research successful cancer treatments for many cancers is still an elusive goal. What are needed are new therapeutics that can attack and eliminate a cancer. SUMMARY Disclosed are bi-specific antibodies that bind an oncogene-binding domain and an α-PIGR binding domain. As disclosed herein, the α-PIGR binding domain facilitates translocation of the antibody from the cell membrane into the cytosol. Binding of the antibody to PIGR on the cell surface causes the antibody to be trafficked in endosomes through a process termed transcytosis, until it reaches the cell membrane on a different pole, where a fragment of PIGR is cleaved. This results in a complex consisting of the bi-specific antibody, a fragment of PIGR (now termed the secretory component), and any antigen they encountered through transcytosis. Therefore, disclosed herein is a bispecific antibody having an oncogene or gene fusion-binding domain and an α-PIGR binding domain. In some embodiments, Attorney Docket Number: 10110-425WO1 the α-PIGR binding domain comprises an α-PIGR-binding variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and an α-PIGR-binding variable light (VL) domain having CDR1, CDR2 and CDR3 sequences. In some embodiments, the CDR1 sequence of the α-PIGR-binding VH domain comprises the amino acid sequence GYTFIEYT (SEQ ID NO:1); the CDR2 sequence of the α-PIGR-binding VH domain comprises the amino acid sequence INPNNGGT (SEQ ID NO:2); the CDR3 sequence of the α-PIGR-binding VH domain comprises the amino acid sequence ARYYRYDVLSAMDY (SEQ ID NO:3); the CDR1 sequence of the α-PIGR-binding VL comprises the amino acid sequence ESVDNYAISF (SEQ ID NO:4); the CDR2 sequence of the α-PIGR-binding VL domain comprises the amino acid sequence AAS; and the CDR3 sequence of the α-PIGR-binding VL domain comprises the amino acid sequence QQSKAVPYT (SEQ ID NO:5). Therefore, in some embodiments, the α-PIGR-binding VH domain comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFIEYTMHWVRQAPGQGLEWMGWINPN NGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARYYRYDVLSAMD YWGQGTLVTVSS (SEQ ID NO:6), and in some embodiments, the α-PIGR-binding VL domain comprises the amino acid sequence EIVLTQSPATLSLSPGERATLSCRASESVDNYAISFLAWYQQKPGQAPRLLIYAASN RATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQSKAVPYTFGQGTKLEIK (SEQ ID NO:7). In some embodiments, the oncogene is α-KRASG12D. The bispecific antibody of claim 4, wherein the oncogene-binding domain comprises a oncogene-binding variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a oncogene-binding variable light (VL) domain having CDR1, CDR2 and CDR3 sequences. In some embodiments, the CDR1 sequence of the oncogene-binding VH domain comprises the amino acid sequence GTFSSYA (SEQ ID NO:8); the CDR2 sequence of the oncogene-binding VH domain comprises the amino acid sequence ISRSGHST (SEQ ID NO:9); the CDR3 sequence of the oncogene-binding VH domain comprises the amino acid sequence AKRFGSIVFDY (SEQ ID NO:10); the CDR1 sequence of the oncogene-binding VL comprises the amino acid sequence QSLFNSRTRKNY (SEQ ID NO:11); the CDR2 sequence of the oncogene-binding VL domain comprises the amino acid sequence WAS; and the CDR3 sequence of the oncogene-binding VL domain comprises the amino acid sequence KQSYYHMYT (SEQ ID NO:12). Therefore, in some embodiments, the α oncogene-binding VH Attorney Docket Number: 10110-425WO1 domain comprises the amino acid sequence MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGTFSSYAMSWV RQAPGKGLEWVSTISRSGHSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCAKRFGSIVFDYWGQGTLVTVSS (SEQ ID NO:13), and in some embodiments the oncogene-binding VL domain comprises the amino acid sequence MGWSCIILFLVATATGVHSDLVMTQSPATLSLSPGERATLSCKSSQSLFNSRTRKNY LAWYQQKPGQAPRLLIYWASTRESGIPGRFSGSGSGTDFLTISSLEPEDFAVYYCK QSYYHMYTFGQGTKVEIK (SEQ ID NO:14). In some embodiments, the bi-specific antibody has the following formula: VLO – VHO – VLP – VHP, VLP – VHP – VLO – VHO, VHP – VLP – VHO – VLO, VHO – VLO – VHP – VLP, VLO – VHO – VHP – VLP, or VLP – VHP – VHO – VLO, wherein “VHO” is an oncogene or gene fusion-binding VH domain; wherein “VLO” is an oncogene or gene fusion-binding VL domain; wherein “VHP” is a α-PIGR-binding VH domain; wherein “VLP” is a α-PIGR-binding VL domain; and wherein “–” consists of a peptide linker or a peptide bond. In some embodiments, the has a first polypeptide, second polypeptide, third polypeptide, and fourth polypeptide, wherein the first polypeptide and second polypeptide have the formula: VHO – CH1 = CH2 – CH3 – VLP – VHP, or VHO – CH1 = CH2 – CH3 – VHP – VLP; wherein the third polypeptide and fourth polypeptide have the formula VLO – CL; wherein “CH1” is a first heavy chain constant domain; wherein “CH1” is a second heavy chain constant domain; wherein “CH1” is a third heavy chain constant domain; wherein “CL” is a light chain constant domain; wherein “VHO” is an oncogene or gene fusion-binding VH domain; wherein “VLO” is an oncogene or gene fusion -binding VL domain; wherein “VHP” is a α-PIGR-binding VH domain; Attorney Docket Number: 10110-425WO1 wherein “VLP” is a α-PIGR-binding VL domain; wherein “–” consists of a peptide linker or a peptide bond; wherein “=” is a heavy chain hinge domain; and wherein the first polypeptide and second polypeptide are conjugated to each other at their hinge domains, wherein the third polypeptide is conjugated to the first polypeptide at their CL and CH1 domains, respectively, and wherein the fourth polypeptide is conjugated to the second polypeptide at their CL and CH1 domains, respectively. In some embodiments, the oncogene is selected from the group consisting of ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM, BCLAF1, BRAF, BRCA1, BRCA2, CCND3, CTNNB1, CREBBP, DNMT3A, EGFR, EP300, ERBB2, ERBB3, ESR1, EZH2, FAT1, FAT3, FAT4, FBXO11, FBXW7, FGFR1, FLT3, FOXA1, GNA11, GNAQ, GNAS, GTF2I, H3F3A, HER2/NEU, HRAS, IDH1, IDH2, JAK2, KCNJ4, KDM6A, KIT, KMT2C, KMT2D, KRAS, LRP1B, MET, MTOR, MYC, MYCN, NF1, NOTCH1, NRAS, NSD1, NSD2, NTRK3, OBSCN, PCBP1, PIK3CA, PIK3R1, PIGR, PLCG1, PTCH1, PTEN, PTPN6, PTPN11, PTPN13, RAC1, RB1, RET, ROS1, RHOA, RYR2, SET2D, SF3B1, SMAD2, SMAD4, SMARCA4, SRC, SRSF2, TP53, TRRAP, TTN, U2AF1L4, VHL, and CDKN2A. In some aspects the gene fusion is selected from the group consisting of EML4-ALK, KIF5B-ALK, HIP1-ALK, KLC1-ALK, DCTN1-ALK, PRKAR1A-ALK, STRN-ALK, CLTC-ALK, MPRIP-ALK, NPM1-ALK, TNS1-ALK, ACTG2-ALK, IGFBP5- ALK, SEC31A-ALK, TPM3-ALK, AITC-ALK, TPM4-ALK, CD74 ROS1, SLC34A2- ROS1, SDC4-ROS1, EZR-ROS1, LR1G3-ROS1, SDC4-ROS1, TPM3-ROS1, LIMA1- ROS1, MSN-ROS1, WNK1-ROS1, RBPMS-ROS1, KIF5B-RET, CCDC6-RET, and TRIM33-RET. Also disclosed is a method of treating a cancer in a subject comprising administering to the subject a bispecific antibody disclosed herein. Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis associated with expression of an oncogene (such as, for example, ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM, BCLAF1, BRAF, BRCA1, BRCA2, CCND3, CTNNB1, CREBBP, DNMT3A, EGFR, EP300, ERBB2, ERBB3, ESR1, EZH2, FAT1, FAT3, FAT4, FBXO11, FBXW7, FGFR1, FLT3, FOXA1, GNA11, GNAQ, GNAS, GTF2I, H3F3A, HER2/NEU, HRAS, IDH1, IDH2, JAK2, KCNJ4, KDM6A, KIT, KMT2C, KMT2D, KRAS, LRP1B, Attorney Docket Number: 10110-425WO1 MET, MTOR, MYC, MYCN, NF1, NOTCH1, NRAS, NSD1, NSD2, NTRK3, OBSCN, PCBP1, PIK3CA, PIK3R1, PIGR, PLCG1, PTCH1, PTEN, PTPN6, PTPN11, PTPN13, RAC1, RB1, RET, ROS1, RHOA, RYR2, SET2D, SF3B1, SMAD2, SMAD4, SMARCA4, SRC, SRSF2, TP53, TRRAP, TTN, U2AF1L4, VHL, and CDKN2A), gene overexpression, or a gene fusion (such as, for example, ALK fusions including, but not limited to EML4-ALK, KIF5B-ALK, HIP1-ALK, KLC1-ALK, DCTN1-ALK, PRKAR1A-ALK, STRN-ALK, CLTC-ALK, MPRIP-ALK, NPM1-ALK, TNS1-ALK, ACTG2-ALK, IGFBP5-ALK, SEC31A-ALK, TPM3-ALK, AITC-ALK, TPM4-ALK; ROS1 fusions including, but not limited to, CD74 ROS1, SLC34A2-ROS1, SDC4-ROS1, EZR-ROS1, LR1G3-ROS1, SDC4-ROS1, TPM3-ROS1, LIMA1-ROS1, MSN-ROS1, WNK1-ROS1, RBPMS-ROS1; or RET fusions including, but not limited to, KIF5B- RET, CCDC6-RET, and TRIM33-RET) in a subject comprising administering to the subject the antigen binding molecule of any preceding aspect (including, but not limited to a dimeric IgA antigen binding 5 molecule that targets KRASG12D). In some aspects, the dimeric antigen molecule can be administered in one or more viral vectors and self-assemble inside the cancerous cell. For example, disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis associated with expression of an oncogene (such as, for example, 10 ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM, BCLAF1, BRAF, BRCA1, BRCA2, CCND3, CTNNB1, CREBBP, DNMT3A, EGFR, EP300, ERBB2, ERBB3, ESR1, EZH2, FAT1, FAT3, FAT4, FBXO11, FBXW7, FGFR1, FLT3, FOXA1, GNA11, GNAQ, GNAS, GTF2I, H3F3A, HER2/NEU, HRAS, IDH1, IDH2, JAK2, KCNJ4, KDM6A, KIT, KMT2C, KMT2D, KRAS, LRP1B, MET, MTOR, MYC, MYCN, NF1, NOTCH1, NRAS, NSD1, NSD2, NTRK3, OBSCN, PCBP1, PIK3CA, PIK3R1, PIGR, PLCG1, PTCH1, PTEN, PTPN6, PTPN11, PTPN13, RAC1, RB1, RET, ROS1, RHOA, RYR2, SET2D, SF3B1, SMAD2, SMAD4, SMARCA4, SRC, SRSF2, TP53, TRRAP, TTN, U2AF1L4, VHL, and CDKN2A) , gene overexpression, or a gene fusion (such as, for example, ALK fusions including, but not limited to EML4-ALK, KIF5B-ALK, HIP1-ALK, KLC1-ALK, DCTN1-ALK, PRKAR1A-ALK, STRN-ALK, CLTC-ALK, MPRIP-ALK, NPM1-ALK, TNS1-ALK, ACTG2-ALK, IGFBP5-ALK, SEC31A-ALK, TPM3-ALK, AITC-ALK, TPM4-ALK; ROS1 fusions including, but not limited to, CD74 ROS1, SLC34A2- ROS1, SDC4-ROS1, EZR-ROS1, LR1G3-ROS1, SDC4-ROS1, TPM3-ROS1, LIMA1- ROS1, MSN-ROS1, WNK1-ROS1, RBPMS-ROS1; or RET fusions including, but not Attorney Docket Number: 10110-425WO1 limited to, KIF5B-RET, CCDC6-RET, and TRIM33-RET) in a subject comprising administering to the subject 3 viral vectors (such as, for example, pBMN, adeno- associated virus (AAV), Adenovirus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus (HIV), Sindbis virus, and Murine Maloney Leukemia virus) each comprising different antibody constructs; wherein a first viral construct encodes an antibody J chain; a second viral construct encodes an antibody VH chain, and a third viral construct encodes an antibody VL chain; wherein expression of the VH, VL, and J chains causes self-assembly of a dimeric antigen binding molecule; wherein the dimeric antigen binding molecule binds to the oncogene in the cytosol; and wherein the antigen binding molecule is secreted out of the cell while still captured to the bound the oncogene. In one aspect, disclosed herein are methods of making a dimeric antigen binding molecule comprising transfecting a cell with 3 viral vectors (such as, for example, pBMN, adeno-associated virus (AAV), Adenovirus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus (HIV), Sindbis virus, and Murine Maloney Leukemia virus) each comprising different antibody constructs; wherein a first viral construct encodes an antibody J chain; a second viral construct encodes an antibody VH chain, and a third viral construct encodes an antibody VL chain; wherein expression of the VH, VL, and J chains causes formation of a dimeric antigen binding molecule. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. DESCRIPTION OF DRAWINGS Figure 1 illustrates embodiments of the disclosed bispecific antibodies. Figures 2A, 2B, 2C, 2D, 2E, 2F, and 2G show KRASG12D mutation-specific dIgA1, but not IgG4, neutralizes KRASG12D inside tumor cells and expels the oncodriver outside tumor cells. Figure 2A shows immunoblots showing that recombinant anti-KRASG12D-IgA1 and anti-KRASG12D-IgG4 antibodies specifically recognize this mutation in KRAS, but not G12C or G13D mutant KRAS, or WT KRAS (n=3). Figure 2B shows confocal immunofluorescence microscopy images showing that Alexa Fluor 647-conjugated anti-KRASG12D-IgA1, but not with anti-KRASG12D- IgG4, penetrates KRASG12D-mutant A427 and SK-LU-1 NSCLC cells, while KRASG12D-specific, but not control irrelevant dIgA, co-immunoprecipitated with Attorney Docket Number: 10110-425WO1 KRASG12D; Scale bar, 25 μm. Figure 2C shows that OVCAR3 cells were transduced with KRASG12D or WT KRAs fused to PAmCherry (Red). Confocal microscopy shows that treatment with Alexa Fluor 488-conjugated anti-KRASG12D-IgA1 (middle panel), but not with anti-KRASG12D-IgG4 (right panel), disarrays KRASG12D intracellular distribution, without effects on KRASWT cells (left panel). (n=3). Scale bar, 50 μm Figure 2D shows the secretory component of human PIGR was immunoprecipitated from media from KRASG12D-PAmCherry-transduced OVCAR3 cells, treated with anti-KRASG12D-IgA1, IgG4, irrelevant IgA or PBS vehicle. Eluted immunocomplexes were ran by SDS-PAGE, stained with Ponceau Red, and immunoblotted for KRASG12D, IgA, and pIgR (n=3). Figure 2E shows immunoblots showing KRASG12D levels in anti-KRASG12D-IgA1/IgG4 or irrelevant IgA-treated KRASG12D-OVCAR3 cell lysates. Figure 2F shows supernatants from KRASG12D- PAmCherry-transduced OVCAR3 cells, treated with anti-KRASG12D-IgA1, IgG4, irrelevant IgA or PBS vehicle, were subjected to liquid chromatography with tandem mass spectrometry (LC–MS/MS). Heatmaps showing intensities of mCherry (left panel) and pIgR (right panel) peptides detected (n=3). Figure 2G shows Left, Transcytosis experiment adding biotinylated anti-KRASG12D-IgA1/IgG4 or irrelevant IgA to the upper chamber of a transwell system, where physical access of antibodies to the basal chamber is prevented by WT or KRASG12D-OVCAR3 cells grown to confluence in transwell inserts. Right, dot blots showing presence of KRASG12D (top panel) or beads (bottom panel) in the streptavidin immunoprecipitates of the basal and upper chamber contents. Figures 3A and 3B show KRASG12D-specific dIgA1, but not the same antibody on an IgG4 backbone, captures mutant KRAS during trafficking. Figure 3A shows high resolution confocal microscopy showing that aggregates of KRASG12D-specific dIgA, but not KRASG12D-specific IgG, co-localize with KRASG12D-PamCherry after 1 hr of treatment inside KRASG12D-OVCAR3 tumor cells (n=3). No-wash images confirm equivalent positive green fluorescence of added KRASG12D-specific IgA and IgG antibodies outside of the cells (45 min after treatment), Scale bar, 50 μm or 10 μm, as indicated. Figure 3B shows Top panel, Non-denatured lysates (6 mg) of KRASG12D-OVCAR3 cells treated with anti-KRASG12D-IgA1, anti-KRASG12D-IgG4, or vehicle (PBS) were immunoprecipitated using anti-Rab5A, Rab7A, Rab8A, or Rab11A antibodies. Immunoprecipitates were then blotted for KRASG12D-specific IgA or IgG. Bottom panel, 60 µg lysates (1% of co-IP lysate) of anti-KRASG12D-IgA1, anti- Attorney Docket Number: 10110-425WO1 KRASG12D-IgG4, or vehicle (PBS)-treated KRASG12D-OVCAR3 cells were immunoblotted for Rab5A, Rab7A, Rab8A, and Rab11A as input control. (n=3). Figures 4A, 4B, 4C, 4D, 4E, and 4F show KRASG12D-specific dIgA treatment reduces proliferation, without causing apoptosis, of KRASG12D-mutated, but not KRASWT cancer cells. Figure 4A shows MTT assay-based absorbance at 570 nm of KRASG12D-transduced OVCAR3 cells treated with irrelevant IgA or anti-KRASG12D IgA1, relative to mean absorbance of vehicle (PBS) treated cells, at different temporal points. (n=8) Figure 4B shows MTT assay-based absorbance at 570 nm of KRASWT-transduced OVCAR3 cells treated with irrelevant IgA or anti-KRASG12D IgA1, relative to mean absorbance of vehicle (PBS) treated cells, at different temporal points. (n=8). Figure 4C shows MTT assay-based absorbance at 570 nm of untransduced OVCAR3 cells treated with irrelevant IgA or anti-KRASG12D IgA1, relative to mean absorbance of vehicle (PBS) treated cells, at different temporal points. (n=16). Figure 4D shows MTT assay-based absorbance at 570 nm of KRASG12D SK-LU-1 cells treated with irrelevant IgA or anti-KRASG12D IgA1, relative to mean absorbance of vehicle (PBS) treated cells, at different temporal points. (n=16). Figure 4E shows MTT assay-based absorbance at 570 nm of PIGR- HEK293T cells treated with irrelevant IgA or anti-KRASG12D IgA1, relative to mean absorbance of vehicle (PBS) treated cells, at different temporal points. (n=16). Figure 4F shows the total apoptotic cells, determined by flow cytometry analysis of KRASG12D-transduced OVCAR3 cells, treated with vehicle, or irrelevant IgA, or anti-KRASG12D IgA1, or anti- KRASG12D IgA4 antibodies, and gated on Annexin V+ Propidium iodide+/- cells (n=3). Data are mean ± SEM. ***P ≤ 0.001, NS, not significant; unpaired two-tailed t-test. Figures 5A, 5B, 5C, 5D, and 5E show KRASG12D-specific dIgA1, but not the same antibody on an IgG4 backbone, abrogates KRASG12D tumor growth in vivo. Figure 5A shows a schematic of design of experiment shown in B and C. Antibody (100 μg per 20 g body weight) or equal volume of vehicle (PBS) was intratumorally (IT) injected. Figure 5B shows tumor growth curves (left), tumor weight (right) in KRASG12D-OVCAR3 tumor-bearing Rag1-deficient mice receiving control IgA, anti- KRASG12D-IgA1, anti-KRASG12D-IgG4 antibodies, or vehicle. Growth curves and tumor weights were pooled from 2 independent experiments (n=10 mice per group, total). Figure 5C shows tumor growth curves (left), tumor weight (right) from KRASWT- OVCAR3 tumor-bearing Rag1-deficient mice receiving the same treatments as in B. Growth curves and tumor weights were pooled from 2 independent experiments Attorney Docket Number: 10110-425WO1 (n=10 mice per group, total). Figure 5D shows a schematic of design of experiment. Antibody (100 μg per 20 g body weight), equal volume of vehicle (PBS), or MRTX1133 (200 μg per 20 g body weight) were intraperitoneally (IP) injected every 4 days, or daily, as indicated, into NSG mice growing KRASG12D-OVCAR3 tumors. Tumor growth curves (left) and tumor weight (right) were pooled from 3 independent experiments (n=13 mice per group, total). Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, NS, not significant; paired two-tailed t-test for growth curves or unpaired two-tailed t-test for tumor weights. Figure 5E shows a representative immunohistochemical staining of human IgA of KRASG12D-OVCAR3 mice tumors treated intraperitoneally with non-antigen specific or KRASG12D-specific dIgA. Scale bar, 400 or 60 µm, as indicated. Figures 6A, 6B, 6C, 6D, 6E, 6F, and 6G show KRASG12D-specific dIgA1 is more effective than small molecule KRASG12D inhibitors at abrogating the progression of KRASG12D NSCLC. Figure 6A shows the combined staining of pIgR, IgA, IgG, CD3, Pan cytokeratin (panCK) and DAPI, representative of 30 clinical cases of squamous cell carcinoma or adenocarcinoma of the lung. Scale bar, 200 μm (upper panel), 100 μm (magnified lower panels). Figure 6B shows the accumulation of CD3+ T cells in human panCK+ NSCLC islets are associated with increased coating of tumor cells with IgA and IgG, expression of pIgR and co-localization of IgA with pIgR. Data are mean ± SEM. **P≤0.01; ***P≤0.001, unpaired two-tailed t-test. Figure 6C shows a schematic of design of experiment shown in D&E. Antibody (100 μg per 20 g body weight), or equal volume of vehicle (PBS) were administered every 4 days, while MRTX1133 (200 μg per 20 g body weight) was administered daily or every 4 days, as indicated. All treatments were IP injected. Figure 6D shows tumor growth curves from KRASG12D A427 tumor-bearing NSG mice receiving (IP) anti-KRASG12D-IgA1, anti-KRASG12D-IgG4 or vehicle every 4 days. MRTX1133 was administered in different mice every 4 days (left) or daily (middle), as indicated. Tumor weight is shown on the right. Growth curves and tumor weights were pooled from 3 independent experiments (n=14 mice per group, total). Figure 6E shows Western blot showing phosphorylated and total ERK1/2 in lysates of KRASG12D-mutated A427 cells, treated with anti-KRASG12D-IgA1 or MRTX1133. Figure 6F shows identical treatment as in ‘D’ of advanced KRASG12D-mutated SK-LU-1 lung cancers. Growth curves and tumor weights were pooled from 3 independent experiments (n=14 mice per group, total). Figure 6G shows Left, schematic design of treatments in Attorney Docket Number: 10110-425WO1 KPMSH2KIN lung tumor-bearing immunocompetent mice. Antibody (100 μg per 20 g body weight) or equal volume of vehicle (PBS) was IP injected. When indicated, CD8 T cells were depleted with intraperitoneal anti-CD8 antibodies. Right, Tumor growth curves from mice receiving intraperitoneal vehicle or anti-KRASG12D-dIgA1, or anti- IDH1R132H-dIgA1, with or without CD8 T cell depletion. Growth curves were pooled from 2 independent experiments (n=8-10 mice per group). Data are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; paired two-tailed t-test (D&F) and non-parametric Wilcoxon test (G) for growth curves or unpaired two-tailed t-test for tumor weights. Figures 7A, 7B, 7C, 7D, 7E, 7F, and 7G show IDH1R132H-specific dIgA1 abrogates IDH1-mutated colon cancer growth in vivo in a PIGR expression- dependent manner. Figure 7A shows immunoblots showing that recombinant anti- IDH1R132H-IgA1 antibodies specifically recognize the mutation in the lysates of IDH1R132H/+ HCT116 cells, but not in IDH1+/+ HCT116 cells, with or without PIGR- transduction. Overexpression of PIGR protein in PIGR-transduced cells is also shown (n=3). Figure 7Bs shows a schematic of design of experiment shown in C-F. Antibody (100 μg per 20 g body weight) or equal volume of vehicle (PBS) was injected IP. Figure 7C shows untransduced (PIGRlow) mutant-IDH1 (IDH1+/R132H) tumor-bearing NSG mice received IP control IgA, anti-IDH1G12D-IgA1, or vehicle. Tumor growth curves (left), tumor weight (middle) and tumor volume of one representative experiment (right) are shown. Growth curves and tumor weights were pooled from 3 independent experiments (n=14 mice per group, total). Figure 7D shows tumor growth curves (left), tumor weight (middle) and tumor volume of one representative experiment (right) from identically treated untransduced (PIGRlow) WT- IDH1 (IDH1+/+) tumor-bearing NSG mice. Growth curves and tumor weights were pooled from 3 independent experiments (n=15 mice per group, total). Figure 7E shows tumor growth curves (left), tumor weight (middle), tumor volume of one representative experiment (right) from PIGR-transduced mutant-IDH1 (IDH1+/R132H) tumor-bearing NSG mice treated as in C&D. Growth curves and tumor weights were pooled from 3 independent experiments (n=16 mice per group, total). Figure 7F shows tumor growth curves (left), tumor weight (middle), tumor volume of one representative experiment (right) from PIGR-transduced WT-IDH1 (IDH1+/+) tumor- bearing NSG mice treated as in 7C-7E. Growth curves and tumor weights were pooled from 3 independent experiments (n=16 mice per group, total). Figure 7G shows a schematic of design of experiment. Monomeric or dimeric anti-IDH1G12D- Attorney Docket Number: 10110-425WO1 IgA1, anti-IDH1G12D-IgG4 antibody or equal volume of vehicle (PBS) were injected IP into mutant-IDH1 (IDH1+/R132H) tumor-bearing NSG mice. All antibodies were administered at100 μg per 20 g body weight. Growth curves pooled from 2 independent experiments (n=8-9 mice per group, total). Data are mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001, NS, not significant; paired two-tailed t-test for growth curves or unpaired two-tailed t-test for tumor weights. Figures 8A, 8B, and 8C show PIGR mRNA expression across cancer types and confirmation of dimeric fraction in native IgA antibodies. Figure 8A shows TCGA data showing PIGR mRNA expression, expressed as RNA Seq V2 RSEM (log2(value+1)) in several epithelial and non-epithelial malignancies. (Figure 8B shows native human IgA was subjected to mass spectrometry. Two unique peptides, one is from amino acid 47 to 58 (SSEDPNEDIVER) and the other from amino acid 61-69 (IIVPLNNR), of human J-chain were identified. Figure 8C shows native gel electrophoresis of two different lot of native human IgA procured from Abcam (ab91025) which shows the presence of both dimeric and monomeric IgA in the gel at their corresponding molecular weights. Figures 9A, 9B, 9C, 9D, 9E, and 9F show production and purification of KRASG12D and IDH1R132H mutation specific recombinant dIgA1 and IgG4 antibodies. Figure 9A shows immunoblots using denatured lysates of transduced HEK293T cells confirming expression of alpha (α) or gamma (γ) heavy chain, kappa (κ) light chain, specific for KRASG12D or IDH1R132H dIgA1 or IgG4, and J-chain for dimeric IgA1 (n=3). Figure 9B shows graphs showing peaks of purified anti-human KRASG12D and IDH1R132H dIgA and IgG4 antibodies at 280nm. Figure 9C shows native gel electrophoresis of different IgA antibodies that shows the presence of dimeric (black arrows) and monomeric IgA (red arrows) in the gel at their corresponding molecular weights. Figure 9D shows immunoblots showing that recombinant anti-KRASG12D- IgA1 and anti- KRASG12D-IgG4 antibodies specifically recognize mutant KRASG12D, in the lysates of KRASG12D-PAmCherry-OVCAR3, but not KRASWT-PAmCherry- OVCAR3 cells, as well as KRASG12D mutated A427 and SK-LU-1 lung cancer cell lines. Anti-mCherry antibody recognized KRASWT-PAmCherry and KRASG12D- PAmCherry fusion proteins in transduced OVCAR3 lysates (n=3). Figure 9E shows immunoblots showing KRASG12D levels in anti-KRASG12D-IgA1/IgG4 or non-antigen specific irrelevant IgA-treated KRASG12D-A427 lung cancer cell lysates. Figure 9F shows transcytosis experiment adding biotinylated anti-KRASG12D-IgA1/IgG4 or Attorney Docket Number: 10110-425WO1 irrelevant IgA to the upper chamber of a transwell system, where physical access of antibodies to the basal chamber is prevented by WT or KRASG12D-OVCAR3 cells grown to confluence in transwell inserts. Dot blots showing presence of KRASG12D (left panel), human IgA (middle panel), or human IgG (right panel) in the streptavidin immunoprecipitates of the basal and upper chamber contents. Figures 10A, 10B, and 10C show intratumoral infusion of KRASG12D-specific dIgA1, but not the same antibody on an IgG4 backbone abrogates KRASG12D- OVCAR3, but not KRASWT-OVCAR3 tumor growth, superior to control IgA and MRTX1133, in Rag1-deficient mice. Figure 10A schematic of design of experiment shown in ‘B’, and ‘C’. Antibody (100 μg per 20 g body weight) or equal volume of vehicle (PBS) was intratumorally (IT) injected every 4 d, or MRTX1133 (200 μg per 20 g body weight) were injected intraperitoneally (IP) every 4 d or daily. Figure 10B tumor growth curves (left), tumor weight (middle), tumor volume (right) in KRASG12D- OVCAR3 tumor-bearing Rag1-deficient mice. Growth curves and tumor weights were pooled from 2 independent experiments (n=10 mice per group, total). Figure 10C tumor growth curves (left), tumor weight (middle), tumor volume (right) in KRASWT- OVCAR3 tumor-bearing Rag1-deficient mice. Growth curves and tumor weights were pooled from 2 independent experiments (n=10 mice per group, total). Data are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, NS, not significant; paired two-tailed t-test for growth curves or unpaired two-tailed t-test for tumor weights. Figures 11A and 11B show intraperitoneal infusion of KRASG12D-specific dIgA1, but not the same antibody on an IgG4 backbone abrogates KRASG12D- OVCAR3, but not KRASWT- OVCAR3 tumor growth, superior to control IgA and MRTX1133, in NSG mice. Figure 11A shows a schematic of design of experiment shown in ‘B’. Antibody (100 μg per 20 g body weight) or equal volume of vehicle (PBS) every 4 d or MRTX1133 (200 μg per 20 g body weight) every 4 d or daily were injected intraperitoneally (IP). Figure 11B shows tumor growth curves (top), tumor weight (bottom left), representative tumor volume of one experiment (bottom right) in KRASG12D-OVCAR3 tumor- bearing NSG mice. Growth curves and tumor weights were pooled from 3 independent experiments (n=13 mice per group, total). Data are mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001, NS, not significant; paired two-tailed t-test for growth curves or unpaired two-tailed t-test for tumor weights. Figures 12A, 12B, 12C, 12D, AND 12E show intraperitoneal infusion of KRASG12D-specific dIgA1, but not the same antibody on an IgG4 backbone abrogates Attorney Docket Number: 10110-425WO1 KRASG12D mutated A427 or SK-LU-1 tumors, superior to control IgA and MRTX1133, in NSG mice. Figure 12A shows bar graphs showing comparisons of percentages of CD3+ cells, PIGR+ cells, IgA-coated cells, and IgG-coated cells within the PCK+ tumor islets among adenocarcinoma and squamous cell carcinoma histology types. (n=12 each, with two duplicated cores). NS, not significant; Unpaired two-tailed t-test. Figure 12B shows a schematic of design of experiment shown in ‘C’ and ‘D’. Antibody (100 μg per 20 g body weight) or equal volume of vehicle (PBS) every 4 d or MRTX1133 (200 μg per 20 g body weight) daily or every 4 d were injected IP. Figure 12C shows tumor growth curves (top), tumor weight (bottom, left), tumor volume (bottom, right) in A427 tumor-bearing NSG mice. Growth curves and tumor weights were pooled from 3 independent experiments (n=14 mice per group, total). Data are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, NS, not significant; paired two-tailed t- test for growth curves or unpaired two-tailed t-test for tumor weights. Figure 12D shows tumor growth curves (top), tumor weight (bottom, left), tumor volume (bottom, right) in SK-LU-1 tumor-bearing NSG mice. Growth curves and tumor weights were pooled from 3 independent experiments (n=14 mice per group, total). Figure 12E shows Left, schematic design of treatments in KRASG12D Brpkp110 tumor-bearing immunocompetent mice. Antibody (100 μg per 20 g body weight) were intraperitoneally injected. Middle, tumor growth curves and right, tumor weights in mice receiving intraperitoneal anti-KRASG12D-dIgA1, or anti-KRASG12D-IgG4, or non- specific dIgA. Growth curves and tumor weights were pooled from 2 independent experiments (n=10 mice per group, total). Data are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, NS, not significant; paired two-tailed t-test for growth curves or unpaired two- tailed t-test for tumor weights. DETAILED DESCRIPTION Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or Attorney Docket Number: 10110-425WO1 intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts Attorney Docket Number: 10110-425WO1 by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere. Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two Attorney Docket Number: 10110-425WO1 particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings: “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant. A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant. "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be Attorney Docket Number: 10110-425WO1 referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control. By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination. The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific Attorney Docket Number: 10110-425WO1 therapy directed toward the improvement of the associated disease, pathological condition, or disorder. "Biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject. "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of'' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of'' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure. A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative." “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated Attorney Docket Number: 10110-425WO1 into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration. "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein. “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree. “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. Attorney Docket Number: 10110-425WO1 “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years. Throughout this application, various publications are referenced. 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 to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Compositions Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular dimeric antigen binding molecules is disclosed and discussed and a number of modifications that can be made to a number of molecules including the dimeric antigen binding molecules are discussed, specifically contemplated is each Attorney Docket Number: 10110-425WO1 and every combination and permutation of dimeric antigen binding molecules and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods. Antibodies Antibodies Generally In some aspects, the disclosed antigen binding molecules are antibodies. The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with an oncogene such that the oncogene is bound in the cytosol of a cell and secreted outside the cell while being bound to said oncogene. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. In some embodiments, the antibody is not an IgA or IgM antibody. Therefore, in some embodiments, the antibody has an IgG backbone. Attorney Docket Number: 10110-425WO1 The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity. The disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The monoclonal antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No.5,804,440 to Burton et al. and U.S. Patent No.6,096,441 to Barbas et al. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec.22, 1994 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a Attorney Docket Number: 10110-425WO1 residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen. As used herein, the term “antibody or fragments thereof” encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab, Fv, scFv, VHH, and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain oncogene (such as KRASG12D) binding activity are included within the meaning of the term “antibody or fragment thereof.” Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)). Also included within the meaning of “antibody or fragments thereof” are conjugates of antibody fragments and antigen binding proteins (single chain antibodies). The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non- modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992). As used herein, the term “antibody” or “antibodies” can also refer to a human antibody and/or a humanized antibody. Many non-human antibodies (e.g., those Attorney Docket Number: 10110-425WO1 derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response. Human antibodies Human antibodies can be prepared using any technique. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein. Humanized antibodies Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an sFv, Fv, Fab, Fab’, F(ab’)2, or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non- human (donor) antibody integrated into the framework of a human (recipient) antibody. To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non- Attorney Docket Number: 10110-425WO1 human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)). Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Patent No.4,816,567 (Cabilly et al.), U.S. Patent No. 5,565,332 (Hoogenboom et al.), U.S. Patent No.5,721,367 (Kay et al.), U.S. Patent No.5,837,243 (Deo et al.), U.S. Patent No.5, 939,598 (Kucherlapati et al.), U.S. Patent No.6,130,364 (Jakobovits et al.), and U.S. Patent No.6,180,377 (Morgan et al.). Administration of antibodies Administration of the antibodies can be done as disclosed herein. Nucleic acid approaches for antibody delivery also exist. The broadly neutralizing antibodies and antibody fragments can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment. The delivery of the nucleic acid can be by any means, as disclosed herein, for example. Homology/identity It is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein is through defining the variants and derivatives in terms of homology to specific known sequencesSpecifically disclosed are variants of these and other genes and proteins Attorney Docket Number: 10110-425WO1 herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level. Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math.2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol.48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection. The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. Sequences Anti α-PIGR antibody In some embodiments, the α-PIGR-binding antibody can comprise a variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a variable light (VL) domain having CDR1, CDR2 and CDR3 sequences. In some embodiments, the CDR1 sequence of the α-PIGR-binding VH domain comprises the amino acid sequence GYTFIEYT (SEQ ID NO:1); the CDR2 sequence of the α-PIGR-binding VH domain comprises the amino acid sequence INPNNGGT (SEQ ID NO:2); the CDR3 sequence of the α-PIGR-binding VH domain comprises the amino acid sequence ARYYRYDVLSAMDY (SEQ ID NO:3); the CDR1 sequence of the α-PIGR-binding VL comprises the amino acid sequence ESVDNYAISF (SEQ ID NO:4); the CDR2 sequence of the α-PIGR-binding VL domain comprises the amino acid sequence AAS; and the CDR3 sequence of the α-PIGR-binding VL domain comprises the amino acid sequence QQSKAVPYT (SEQ ID NO:5). Attorney Docket Number: 10110-425WO1 In some embodiments, the α-PIGR-binding VH domain comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFIEYTMHWVRQAPGQGLEWMGWINPN NGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARYYRYDVLSAMD YWGQGTLVTVSS (SEQ ID NO:6). In some embodiments, the α-PIGR-binding VL domain comprises the amino acid sequence EIVLTQSPATLSLSPGERATLSCRASESVDNYAISFLAWYQQKPGQAPRLLIYAASN RATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQSKAVPYTFGQGTKLEIK (SEQ ID NO:7). The heavy and light chains are preferably separated by a linker. Suitable linkers for scFv antibodies are known in the art. In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO:15), GGGGSGGGGS (SEQ ID NO:16) or GGGGSGGGGSGGGGS (SEQ ID NO:17). The scFv can have the formula NH3-VH-linker-VL-COOH or NH3-VL-linker-VH-COOH. Anti-oncogene antibody In some embodiments, the oncogene is α-KRASG12D. In some embodiments, the oncogene-binding domain comprises a oncogene-binding variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a oncogene-binding variable light (VL) domain having CDR1, CDR2 and CDR3 sequences. In some embodiments, the the CDR1 sequence of the oncogene-binding VH domain comprises the amino acid sequence GTFSSYA (SEQ ID NO:8); the CDR2 sequence of the oncogene- binding VH domain comprises the amino acid sequence ISRSGHST (SEQ ID NO:9); the CDR3 sequence of the oncogene-binding VH domain comprises the amino acid sequence AKRFGSIVFDY (SEQ ID NO:10); the CDR1 sequence of the oncogene- binding VL comprises the amino acid sequence QSLFNSRTRKNY (SEQ ID NO:11); the CDR2 sequence of the oncogene-binding VL domain comprises the amino acid sequence WAS; and the CDR3 sequence of the oncogene-binding VL domain comprises the amino acid sequence KQSYYHMYT (SEQ ID NO:12). In some embodiments, the oncogene-binding VH domain comprises the amino acid sequence MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGTFSSYAMSWV RQAPGKGLEWVSTISRSGHSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCAKRFGSIVFDYWGQGTLVTVSS (SEQ ID NO:13). Attorney Docket Number: 10110-425WO1 In some embodiments, the oncogene-binding VL domain comprises the amino acid sequence MGWSCIILFLVATATGVHSDLVMTQSPATLSLSPGERATLSCKSSQSLFNSRTRKNY LAWYQQKPGQAPRLLIYWASTRESGIPGRFSGSGSGTDFLTISSLEPEDFAVYYCK QSYYHMYTFGQGTKVEIK (SEQ ID NO:14). Pharmaceutical carriers/Delivery of pharmaceutical products As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in Attorney Docket Number: 10110-425WO1 liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No.3,610,795, which is incorporated by reference herein. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). Pharmaceutically Acceptable Carriers The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier. Attorney Docket Number: 10110-425WO1 Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically- acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and Attorney Docket Number: 10110-425WO1 injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual Attorney Docket Number: 10110-425WO1 physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch.22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp.365- 389. A typical daily dosage of the antibody used alone might range from about 1 µ g/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above. Methods of using the compositions The disclosed compositions can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers. Also disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis associated with expression of an oncogene (such as, for example, ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM, BCLAF1, BRAF, BRCA1, BRCA2, CCND3, CTNNB1, CREBBP, DNMT3A, EGFR, EP300, ERBB2, ERBB3, ESR1, EZH2, FAT1, FAT3, FAT4, FBXO11, FBXW7, FGFR1, FLT3, FOXA1, GNA11, GNAQ, GNAS, GTF2I, H3F3A, HER2/NEU, HRAS, IDH1, IDH2, JAK2, KCNJ4, KDM6A, KIT, KMT2C, KMT2D, KRAS, LRP1B, MET, MTOR, MYC, MYCN, NF1, NOTCH1, NRAS, NSD1, NSD2, NTRK3, OBSCN, PCBP1, PIK3CA, PIK3R1, PIGR, PLCG1, PTCH1, PTEN, PTPN6, PTPN11, PTPN13, RAC1, RB1, RET, ROS1, RHOA, RYR2, SET2D, SF3B1, SMAD2, SMAD4, SMARCA4, SRC, SRSF2, TP53, TRRAP, TTN, U2AF1L4, VHL, and CDKN2A), gene overexpression, or a gene fusion (such as, for example, ALK fusions including, but not limited to EML4-ALK, KIF5B-ALK, HIP1-ALK, KLC1-ALK, DCTN1-ALK, PRKAR1A-ALK, STRN-ALK, CLTC-ALK, MPRIP-ALK, NPM1-ALK, TNS1-ALK, ACTG2-ALK, IGFBP5-ALK, SEC31A-ALK, TPM3-ALK, AITC-ALK, TPM4-ALK; ROS1 fusions including, but not limited to, CD74 ROS1, SLC34A2-ROS1, SDC4-ROS1, EZR-ROS1, LR1G3-ROS1, SDC4-ROS1, TPM3-ROS1, LIMA1-ROS1, MSN-ROS1, WNK1-ROS1, RBPMS-ROS1; or RET fusions including, but not limited to, KIF5B- RET, CCDC6-RET, and TRIM33-RET) in a subject comprising administering to the subject the antigen binding molecule of any preceding aspect (including, but not Attorney Docket Number: 10110-425WO1 limited to a dimeric IgA antigen binding molecule that targets KRASG12D). In some aspects, the dimeric antigen molecule can be administered in one or more viral vectors and self-assemble inside the cancerous cell. For example, disclosed herein are methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing a cancer and/or metastasis associated with expression of an oncogene (such as, for example, ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM, BCLAF1, BRAF, BRCA1, BRCA2, CCND3, CTNNB1, CREBBP, DNMT3A, EGFR, EP300, ERBB2, ERBB3, ESR1, EZH2, FAT1, FAT3, FAT4, FBXO11, FBXW7, FGFR1, FLT3, FOXA1, GNA11, GNAQ, GNAS, GTF2I, H3F3A, HER2/NEU, HRAS, IDH1, IDH2, JAK2, KCNJ4, KDM6A, KIT, KMT2C, KMT2D, KRAS, LRP1B, MET, MTOR, MYC, MYCN, NF1, NOTCH1, NRAS, NSD1, NSD2, NTRK3, OBSCN, PCBP1, PIK3CA, PIK3R1, PIGR, PLCG1, PTCH1, PTEN, PTPN6, PTPN11, PTPN13, RAC1, RB1, RET, ROS1, RHOA, RYR2, SET2D, SF3B1, SMAD2, SMAD4, SMARCA4, SRC, SRSF2, TP53, TRRAP, TTN, U2AF1L4, VHL, and CDKN2A), gene overexpression, or a gene fusion (such as, for example, ALK fusions including, but not limited to EML4-ALK, KIF5B-ALK, HIP1-ALK, KLC1-ALK, DCTN1-ALK, PRKAR1A-ALK, STRN-ALK, CLTC-ALK, MPRIP-ALK, NPM1-ALK, TNS1-ALK, ACTG2-ALK, IGFBP5-ALK, SEC31A-ALK, TPM3-ALK, AITC-ALK, TPM4-ALK; ROS1 fusions including, but not limited to, CD74 ROS1, SLC34A2-ROS1, SDC4-ROS1, EZR-ROS1, LR1G3-ROS1, SDC4-ROS1, TPM3-ROS1, LIMA1-ROS1, MSN-ROS1, WNK1-ROS1, RBPMS-ROS1; or RET fusions including, but not limited to, KIF5B- RET, CCDC6-RET, and TRIM33-RET) in a subject comprising administering to the subject 3 viral vectors (such as, for example, pBMN, adeno-associated virus (AAV), Adenovirus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus (HIV), Sindbis virus, and Murine Maloney Leukemia virus) each comprising different antibody constructs; wherein a first viral construct encodes an antibody J chain; a second viral construct encodes an antibody VH chain, and a third viral construct encodes an antibody VL chain; wherein expression of the VH, VL, and J chains causes self-assembly of a dimeric antigen binding molecule; wherein the dimeric antigen binding molecule binds to the oncogene in the cytosol; and wherein the antigen binding molecule is secreted out of the cell while still captured to the bound the oncogene. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder Attorney Docket Number: 10110-425WO1 cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers such as small cell lung cancer and non- small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon cancer, rectal cancer, prostatic cancer, or pancreatic cancer. The disclosed treatments methods can also include the administration any anti- cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin- stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado- Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran for Injection (Melphalan Hydrochloride), Alkeran Tablets (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), Amboclorin Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane),Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, Avastin (Bevacizumab), Avelumab, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Besponsa (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, Bexxar (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, Campath (Alemtuzumab), Camptosar , (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil--Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant, Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Attorney Docket Number: 10110-425WO1 Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Durvalumab, Efudex (Fluorouracil--Topical), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride , EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi) , Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista , (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil-- Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil--Topical), Fluorouracil Injection, Fluorouracil--Topical, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI- BEVACIZUMAB, FOLFIRI- CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel wafer (Carmustine Implant), Glucarpidase, Goserelin Attorney Docket Number: 10110-425WO1 Acetate, Halaven (Eribulin Mesylate), Hemangeol (Propranolol Hydrochloride), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Idhifa (Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado- Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Leustatin (Cladribine), Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Lomustine, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride) , Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin- stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Attorney Docket Number: 10110-425WO1 Maleate), Netupitant and Palonosetron Hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R- CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hycela (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and , Hyaluronidase Human, ,Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, Rydapt (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar 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Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, Vistogard (Uridine Triacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposome), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Yondelis (Trabectedin), Zaltrap (Ziv-Aflibercept), Zarxio (Filgrastim), Zejula (Niraparib Tosylate Monohydrate), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib), and/or Zytiga (Abiraterone Acetate). Treatment methods can include or further include checkpoint inhibitors include, but are not limited to antibodies that block PD-1 (Pembrolizumab, Nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (Ipilimumab (MDX- 010), Tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7- H4, TIM3, LAG-3 (BMS-986016). A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without Attorney Docket Number: 10110-425WO1 departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. Examples: Dimeric IgA specifically disables intracellular mutated oncodrivers Limited by their large size and considered unable to the
Figure imgf000041_0001
development of antibody-based immunotherapies has been transmembrane or extracellular targets. However, most oncodrivers underpinning tumor-promoting pathways are intracellular proteins inaccessible to conventional therapeutic antibodies. Accordingly, considerable efforts have been made to develop small molecules that target these intracellular pathways by inhibiting enzymatic activity or serving as allosteric modulators. Following a 40-year quest to target KRAS mutations, Sotorasib, a specific KRASG12C small molecule inhibitor, has been recently recently approved by the FDA. On the other hand, inhibitors against KRASG12D, the more common mutation in pancreatic and colon cancer, are currently under testing. Small molecule inhibitors that target oncodrivers offer great therapeutic benefits and have made a big impact in oncology. However, the half-life of many small molecules is ~6 hours, which can limit on target interactions. In contrast, the serum half-life of optimized antibodies is >20 days, which offers more sustained neutralization and can thus be less frequently dosed. We have recently shown that the IgA/IgM polymeric immunoglobulin receptor (PIGR) is quasi-universally expressed in human ovarian and endometrial cancer cells, which allows transcytosis of dIgA through these tumor cells. However, it is unclear whether IgA transcytosis also occurs in non-gynecologic tumors. Furthermore, IgA transcytoses through endosomes, which could prevent direct contact with specific antigens inside tumor cells. Even if transcytosing IgA is not “shielded” by endosomes, targeted antigens could not co-localize inside tumor cells. Experimentation with these processes can determine if it is possible for engineered Attorney Docket Number: 10110-425WO1 dIgA to target intracellular molecules, with important therapeutic implications: First, the mechanisms of resistance that invariably follow the administration of small molecule inhibitors could be still sensitive to antibody-mediated neutralization, and vice versa. Second, the half-life of unmodified IgA ~6 days, but can be further optimized to increase stability. Third, dIgA is particularly abundant at mucosal surfaces, suggesting that exogenous dIgA could be safely administered. In this study, we investigated the feasibility and therapeutic potential of targeting common mutated oncodrivers using recombinant dIgA. Our results provide a rationale for developing dIgA-based therapeutics to neutralize various intracellular antigens in human cancer and other diseases. RESULTS Mutation-specific dIgA neutralizes intracellular KRASG12D and expels it outside tumor cells, without effects on KRASWT cells. To investigate the spectrum of human malignancies that can be susceptible to PIGR-mediated dIgA transcytosis, we first analyzed PIGR expression in tumors in TCGA datasets. We found a clear dichotomy between epithelial malignancies and B- cell lymphoma, which express relatively high PIGR mRNA levels; and non-epithelial tumors such as melanoma or glioblastoma, which express lower levels (Fig.8A). To test whether antigen-specific dIgA, and not non-specific dIgA (Fig.8B&8C), can target specific molecules in the cytoplasm of different carcinomas, we first focused on targeting KRASG12D, a mutational hotspot present in >4% of human cancers. Recombinant antibodies generated against GppNHp-bound KRASG12D (equivalent to the oncogenic GTP-bound form) were produced on a dIgA or monomeric IgG4 backbone, as in anti-PD-1 immune checkpoint inhibitors, using identical variable heavy and variable light sequences (Fig.9A-9C). Although we cannot rule out that GppNHp-KRASG12D-specific Abs recognize GTP-bound conformations driven by other mutations, both dIgA and IgG Abs specifically recognized ectopic or endogenous mutant KRASG12D, but not other KRAS mutations, in Western-blot analysis (Fig.2A; Fig.9D). Notably, only mutation-specific dIgA, but not IgG, penetrated KRASG12D-mutated lung cancer cells (Fig.2B, left), where mutation- specific, but not control dIgA, targeted KRAS (Fig.2B, right). Consistently, KRASG12D-specific dIgA penetrated OVCAR3 cells transduced with KRASWT or KRASG12D fused to photoactivatable (PA)m-Cherry fluorescent protein, but only disrupts cytoplasmic localization and distribution of intracellular KRASG12D (Fig.2C). Attorney Docket Number: 10110-425WO1 Notably, mutant KRAS was not only neutralized inside tumor cells, but also expelled outside the cell through transcytosis, as evidenced by co-occurrence of KRASG12D with secretory IgA in the supernatant of KRASG12D-PAmCherry-transduced OVCAR3 cells upon treatment with KRASG12D-specific dIgA (Fig.1D), with decreased intracellular KRASG12D (Fig.2E & Fig9E). Furthermore, PAmCherry fragments, fused to KRASG12D, were only found in the supernatant of tumor cells treated with KRASG12D-specific dIgA in 3 independent experiments (Fig.2F, left). In contrast, KRASG12D was not found in supernatants upon treatment with KRASG12D-specific IgG4, irrelevant IgA containing dimers, or vehicle (Fig.2D&2F, left). As expected, PIGR was identified in the supernatants of cells treated with antigen-specific or control dIgA (Fig.2F, right). Together, these results indicated that tumor cell- penetrating dIgA can indeed target specific mutations in KRAS inside tumor cells, resulting in intracellular decreased levels and expelling the oncodriver outside the tumor cell, without obvious effects in KRAS unmutated epithelial cells. KRASG12D and mutation-specific dIgA are found in the same endosomal compartments upon transcytosis To confirm bona fide unidirectional transcytosis, we used a classical transwell system where tumor cells prevent the access of dIgA delivered to the upper chamber into the basal chamber, unless it transcytoses through PIGR+ tumor cells. As shown in Fig.2G & Fig.9F, we found an accumulation of KRASG12D antigen in the basal chamber when KRASG12D-mutant tumor cells were treated with antigen-specific dIgA, but not in wild-type tumor cells or upon PIGR ablation. However, a frail KRAS signal was detected using a pan-KRAS antibody in the basal chamber of tumor cells with other mutations, indicating frail recognition of the GTP-bound oncogene. Importantly, only traces of antigen were found in the upper chamber, where dIgA was added and found in similar amounts, supporting that KRASG12D-specific dIgA undergoes “true” transcytosis through PIGR+ tumor cells. Accordingly, high-resolution confocal microscopy confirmed that aggregates of KRASG12D-specific dIgA co-localize with KRASG12D-PamCherry inside tumor cells, while the same antibody on an IgG4 backbone cannot penetrate tumor cells or alter the preferential location of mutant KRAS on the cell surface (Fig.3A). To understand the dynamics of interactions between antigen-specific dIgA and mutant KRAS inside tumor cells, we immunoprecipitated early (RAB5A+), late (RAB7A+) and recycling (RAB8A+/RAB11A+) endosomes in tumor cells treated with Attorney Docket Number: 10110-425WO1 KRASG12D-specific dIgA or IgG. As expected, given the lack of penetration into tumor cells, KRASG12D-specific IgG was undetectable in any endosomes, while dIgA transcytosed through early and late endosomes (Fig.3B). Consistent with known recycling pathways that prevent proteasomal degradation, mutant KRAS was primarily found in RAB8A+/RAB11A+ recycling endosomes when tumor cells were treated with vehicle or antigen-specific IgG (Fig.3B). In contrast, treatment with KRASG12D-specific dIgA concentrated mutant KRAS on the same endosomes through which dIgA traffics inside tumor cells (Fig.3B), indicate that transcytosing dIgA encounters the antigen on the cell membrane and hauls it in endosomes, eventually expelling the target through secretory IgA. Although dIgA traffics through endosomes, dIgA retains its capacity to specifically target intracellular antigens; at least those that are located near the cell membrane. Consistent with capture and extracellular disposal of KRASG12D, treatment of KRASG12D-transduced cancer cells with mutation-specific dIgA inhibited tumor cell proliferation, without effects on the same cells transduced with KRASWT or untransduced ovarian cancer cells (Fig.4A-C). As expected, treatment of endogenously mutated KRASG12D lung cancer cells with mutation-specific dIgA also inhibited tumor cell proliferation (Fig.4D), while neither antigen specific or control dIgA showed any effects on the proliferation of non-malignant HEK293T cells (Fig.4E). Of note, antigen-specific dIgA dampens proliferation of KRASG12D cancer cells without causing apoptosis (Fig.4F). Therefore, antigen-specific dIgA can expel oncodrivers outside tumor cells by preventing endosomal recycling, resulting in KRASG12D accumulation in the same endosomal compartments where dIgA traffics. KRASG12D-specific dIgA specifically abrogates the progression of mutant tumors in vivo To demonstrate the effectiveness of KRASG12D-specific dIgA in vivo, we first performed intra-tumoral infusions of different treatments in KRASG12D-transduced OVCAR3 tumor-bearing Rag1-/- mice. Irrelevant dIgA dampens the RAS pathway by upregulating DUSP phosphatases that dephosphorylate ERK1/2, while activating inflammatory pathways associated with IFN and TNF signaling, as well as pro- apoptotic pathways linked to ER stress. Accordingly, non-specific dIgA delayed the growth of established ovarian tumors. However, the anti-tumor effects of KRASG12D- specific dIgA were significantly greater (Fig.5A&5B), with no obvious signs of toxicity in any treated mice. Consistently with its inability to penetrate tumor cells, the same Attorney Docket Number: 10110-425WO1 variable heavy and light chains on an IgG4 backbone only elicit a non-statistically significant trend towards tumor reduction, attributable to neutralization of extracellular vesicles (Fig.5B & Fig.10A&10B). Supporting the specificity of this intervention, no effects on tumor growth were elicited in tumor cells transduced with KRASWT, (Fig.5C & Fig.10A&10C), beyond the non-antigen-specific effects of IgA transcytosis. Tumor growth was dependent on KRASG12D because, as reported2, the KRASG12D-selective inhibitor MRTX1133, currently under preclinical development, also elicited significant delays in tumor growth, although daily intraperitoneal administration (20 injections) were required (Fig.10A-10C). Selective effectiveness of antigen-specific dIgA was recapitulated when antibodies were intraperitoneally administered in KRASG12D tumor-bearing NSG mice (Fig.5D & Fig.11A&11B), ruling out the requirement of functional myeloid cells or NK lymphocytes in this model, while supporting systemic distribution of dIgA. Again, 20 (daily) doses of intraperitoneal MRTX1133 or 5 injections of KRASG12D-specific dIgA both delayed tumor progression beyond the effects of irrelevant IgA (Fig.5D & Fig.11B). Correspondingly, human IgA was identified in resected tumors (Fig.5E), confirming trafficking of antigen-specific dIgA to tumor beds. These experiments supported the potential of tumor cell-penetrating dIgA to specifically target mutated oncodrivers inside carcinoma cells in vivo. Lung tumors quasi-universally express PIGR and are highly sensitive to mutant oncodriver-neutralizing dIgA To demonstrate the capacity of dIgA to target other human tumors that are spontaneously driven by KRASG12D, as opposed to ectopic KRAS expression, we first focused on non-small cell lung cancer (NSCLC), which is frequently driven by mutant KRAS, including KRASG12D. Analyses of 30 human lung cancer tissues including squamous cell carcinomas and adenocarcinomas, showed PIGR expression in all tumors, along with spontaneous production of IgA and IgG at tumor beds bound to tumor epithelial tissues as well as to stroma (Fig.6A), without differences for histological subtypes (Fig.12A). Notably, PIGR and tumor-bound IgA density, IgG coating of tumor cells, or PIGR:IgA interactions were all associated with stronger T cell accumulation (Fig.6B). We therefore aimed to demonstrate the effectiveness of mutation-specific dIgA against lung carcinomas endogenously driven by KRASG12D. As shown in Fig.6C&6D, 5 intraperitoneal injections of KRASG12D-specific dIgA, or 20 daily treatments of MRTX1133 at high dose (200 ^g), effectively abrogated the Attorney Docket Number: 10110-425WO1 progression of established A427 NSCLC, while the same antibody on an IgG4 backbone had negligible effects (Fig.6C&6D; Fig.12B&12C). Accordingly, treatment with both KRASG12D-specific dIgA and MRTX1133 decreased ERK1/2 phosphorylation in A427 cells in a time-dependent manner (Fig.6E). Anti-tumor effects were not limited to this tumor model, because established KRASG12D-mutant SK-LU-1 lung cancers also responded to KRASG12D-specific dIgA with comparable effectiveness, although they were more resistant to MRTX1133 (Fig.6C&6F; Fig.12B&12D). These experiments support the potential of dIgA-mediated intracellular targeting of KRASG12D to abrogate the progression of spontaneously mutated NSCLCs, in a mutation-specific manner and without off-target effects in unmutated epithelial cells. dIgA transcytosis sensitizes tumor cells to T cell-mediated killing4. To define the contribution of this mechanism, we utilized a syngeneic, immunogenic KRASG12D- mutant KPMSH2KIN lung tumors, generated by cloning lung cancer cells from C57/BL6 mice with inducible mutations, activated using intra-nasal adenoviral Cre; plus restoring MSH2 through lentiviral transduction. As expected, dIgA targeting KRASG12D also elicited a significant delay in tumor growth (Fig.6G). Interestingly, anti-tumor effects in this immunogenic system were dependent on CD8 T cells (Fig.6G), highlighting the complexity of a coordinated adaptive immune response. Further supporting the specificity of dIgA for cognate antigen, similar anti-tumor effects were not observed upon administration of dIgA targeting a different mutation (IDH1R132H) (Fig.6G). Corresponding effects were observed in another syngeneic KRASG12D breast cancer model (Brpkp110; Fig.12E). dIgA is effective against mutations located deeper into the cytoplasm of tumor cells, in a PIGR-dependent manner To demonstrate the feasibility of targeting additional intracellular antigens, located deeper into the cytoplasm of tumor cells; and also using dIgA to target other epithelial cancers different from ovarian and lung carcinomas, we next generated dIgA1 or IgG4 specifically targeting the cytoplasmic R132H mutation in isocitrate dehydrogenase 1 (IDH1), a common hotspot in multiple human tumors (Fig.7A; Fig.9A-9C). To further evaluate the role of PIGR abundance for dIgA effectiveness, we procured isogenic IDH1WT and IDH1+/R132H HCT116 colon cancer cells, which express low PIGR levels, and transduced them with PIGR, resulting in expression levels comparable to those found in multiple carcinoma cell lines (Fig.7A). Low PIGR Attorney Docket Number: 10110-425WO1 expression abrogated the non-specific anti-tumor effects of irrelevant IgA in this system (Fig.7B-7D). Similar to KRASG12D targeting, IDH1R132H-specific dIgA delayed tumor growth in mutant tumors, without effect in the absence of IDH1R132H mutations (Fig.7C&7D). Notably, anti-tumor effects were dependent on PIGR expression in tumor cells, because the administration of IDH1R132H-specific dIgA resulted in a 3.7- fold reduction in tumor growth, compared to vehicle, when PIGR-transduced IDH1R132H HCT116 cells were treated, compared to a 1.8-fold reduction in low-PIGR- expressing parental IDH1R132H HCT116 colon tumors (Fig.7B, 7C, & 7E). Importantly, anti-tumor effects were again mutation-specific, because a comparable reduction in tumor growth was observed when mice with IDH1WT tumors were treated with irrelevant IgA or IDH1R132H-specifc dIgA (Fig.7F). Dimerization of mutation-specific IgA is also required because the monomeric version of the same IDH1R132H-specific IgA showed no significant antitumor effects (Fig.7G). Together these results supported targeting carcinomas of multiple histological origins and different mutated oncodrivers, using antigen-specific dIgA. They also supported the feasibility of targeting tumors despite low PIGR expression, albeit with possibly decreased effectiveness. DISCUSSION Collectively, these data demonstrate the feasibility of specifically targeting commonly mutated hotspots in oncogenes driving malignant progression in many human carcinomas, using antigen-specific dIgA that transcytoses through all epithelial cells but only targets mutated malignant cells. The results confirm that PIGR-mediated transcytosis of dIgA also occurs in non-gynecological carcinomas, including lung and colorectal cancers. Given that PIGR is expressed in most epithelial malignancies, but only at lower levels in non- epithelial cancers, this mechanism can be relevant for most human epithelial cancers. PIGR-mediated dIgA transcytosis progresses through endosomal trafficking but their route of trafficking may slightly differ in malignant cells than in non-malignant cells8. Despite trafficking through endosomes, dIgA retains its capacity to specifically target intracellular antigens. Our results show that dIgA transcytosis elicits loss of recycling endosomes carrying oncogenic KRAS and colocalization of antigen and antibody in early and late endosomes. This is consistent with heterotypic fusion of the early endosomes that internalize dIgA with the recycling endosomes bringing back KRAS to the cell surface. However, this activity is not limited to mutated oncodrivers Attorney Docket Number: 10110-425WO1 that are located near the cell membrane, such as KRAS, but also mutated oncogenes such as IDH1, which are located deeply inside the cytoplasm. Novel studies can determine whether other commonly mutated oncogenes such as PI3K or AKT, or immunosuppressive intracellular pathways such as IDO, can be more effectively targeted with dIgA than with small molecule inhibitors. dIgA-mediated targeting, along or in combination with small molecules, can have significant advantages, compared to small molecules: First, our results underscore the specificity of this approach. Second, although the half-life of IgA (~6 days in primates and >15 hrs in mice) is lower than that of IgG, and there can be significant differences in availability of PIGR-binding dimeric IgA vs. the monomeric form, which does not bind to PIGR but can reach the tumor microenvironment, persistence is nevertheless longer than for small molecule inhibitors, as further supported by our study. Moreover, modifications in glycosylation or attaching an albumin-binding domain to the heavy or light chain can increase their half-life. Third, dIgA expels the mutated oncogene outside tumor cells, which is obviously advantageous, compared to temporary neutralization. Finally, the prospect of oral secretory IgA treatment can make this intervention ideal against tumors of the digestive tract. We therefore propose a rationale for using dIgA as a new form of immunotherapy to target intracellular oncodrivers, which opens multiple new avenues to treat otherwise undruggable carcinomas. Although our dIgA treatments did not fully eliminate xenograft tumors, we only administered 5 injections and we only treated established (i.e., palpable) tumors. We do not know the effect of sustained antibody administration, or higher doses. We found a patchy staining for human IgA, which could reflect suboptimal doses of dIgA, but also different transcytosis timing for distinct tumor areas, or mutational heterogeneity. Our data showing therapeutic effectiveness in immunocompetent mice bearing syngeneic immunogenic carcinomas further supports the concept that the interaction between human dIgA and mouse pIgR mirrors the bioavailability of antigen-specific dIgA in vivo, in the presence of PIGR+ healthy epithelial cells that can compete for the antibody. Furthermore, unlike humans, rats and hamsters, mice do not express CD89, which also binds IgA and could be partially responsible for enlarged persistence of IgA in primates, compared to mice, and therefore retain dIgA in circulation for much longer. Attorney Docket Number: 10110-425WO1 Finally, an important issue regarding the future translatability of dIgA is potential toxicity. In support of our approach, oral IgA-IgG treatment has already been safely used to treat children with chronic non-specific diarrhea, and polyclonal antibody preparations containing >20% IgA can be safely administered in patients with severe pneumonia. In addition, mutation-specific dIgA had no effect on wild-type cells, while dimeric IgA is the predominant Ig at mucosal secretions. It is therefore unlikely that IgA administration can cause on-target, off-tumor toxicity. Furthermore, FDA-approved KRASG12C small molecule inhibitors already elicit grade 3-4 toxicity in >11% of patients. METHODS Data and code availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD042914 and PXD044426. This paper does not report original code. The datasets generated during the current study are available from the lead contact upon reasonable request. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Experimental model and subject details Human samples A lung cancer tissue microarray of total 30 cases including squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma and bronchioloalveolar carcinoma with matched metastatic carcinoma and cancer adjacent tissue, with available pathology grade and survival data was procured from US Biomax. Cell lines OVCAR3 (RRID:CVCL_0465), A427 (RRID:CVCL_1055), SK-LU-1 (RRID:CVCL_0629), HEK293T (RRID:CVCL_0063), NCI-H23 (RRID:CVCL_1547), NCI-H647 (RRID:CVCL_1574) cell lines were purchased from ATCC (Manassas, VA). IDH1+/+ and IDH1R132H/+ HCT116 (RRID:CVCL_0291) cell lines were purchased from Horizon discovery. PIGR-ablated OVCAR3 cells was generated in the lab. Brpkp110 was established in the lab and KPMSH2KIN is a new model of lung cancer developed also in our laboratories that carries the same epitope recognized in human G12D-mutated KRAS. Cell lines were routinely cultured in R10 (RPMI-1640, 10% FBS, penicillin (100 IU ml−1), streptomycin (100 μg ml−1), L-glutamine (2 mM), sodium pyruvate (0.5 mM)) media (Thermo). HEK293T cell line was routinely cultured in D10 Attorney Docket Number: 10110-425WO1 (DMEM, 10% FBS, penicillin (100 IU ml−1), streptomycin (100 μg ml−1), L-glutamine (2 mM), sodium pyruvate (0.5 mM)) media (Thermo).Cell lines were routinely tested for negative mycoplasma contamination. Animal models For mouse models, three different strains were used. Female, 4-6 weeks old Rag1-deficient (Rag1-/-) mice (RRID:IMSR_JAX:003729) and female, 7 weeks old C57BL/6J mice (RRID:IMSR_JAX:000664) were purchased from The Jackson Laboratory. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (RRID:IMSR_JAX:005557), originally procured from The Jackson Laboratory, were maintained by the Moffitt Cancer Center animal facility. All animals were maintained by the animal facility of the Moffitt Cancer Center and Duke School of Medicine, housed in cages of up to 5 mice per cage, in a temperature controlled (18-23°C), 40-60% humidity, 12 h light/dark cycle facility. Animal studies were performed in accordance with Institutional Animal Care and Use Committee at the University of South Florida Research Integrity and Compliance department (IACUC protocols#IS00006655 and #IS00009457) and at the Duke University and Division of Laboratory Animal Resources (IACUC protocol# A011‐23‐01) Method details Constructs, and transduction Human PIGR-coding sequence was cloned into pLVX-IRES-ZsGreen1 lentiviral vector (Genscript). Constructs encoding Wild-type or G12D mutated KRAS fused with PAmCherry1 (pLenti-TO/CMV-PAmCherry1-KRAS, G12D or WT) were procured from Dr. Xiaolin Nan Lab, Oregon Health & Science University. The sequence integrity and accuracy of all constructs was assessed and confirmed by sequencing services from GeneWiz. Virus production and transduction Lentiviral particles were generated by co-transfecting HEK 293T/17 (ATCC) with pLVX-IRES-PIGR-ZsGreen, or pLenti-TO/CMV-PAmCherry1-KRAS (G12D mutated KRAS or WT) and packaging/envelope vector pMD2.G (RRID:Addgene_12259) and psPAX2 (RRID:Addgene_12260) using Lipofectamine- 3000 (Invitrogen) and the viral supernatant was harvested 48h after.5×105 HCT116 or OVCAR3 cells were seeded in a 6-well plate with 3mL of the corresponding 0.45 μ filtered viral supernatant with added 10 μg/mL polybrene (Millipore). The plates were spun for 90min at 32°C and 1200g.12h after transduction the medium was changed Attorney Docket Number: 10110-425WO1 to R10 and 48h later the expression of the target gene was verified under microscope for KRAS transductions and selected by FACS-sorted for ZsGreen expression (PIGR). Tumor Models Flank tumors were initiated by injecting 1×107 KRASWT or G12D transduced OVCAR3 or A427; or 5×106 IDH1WT or Mut HCT116 cells or 5×105 Brpkp110 cells; or 1 × 106 KPMSH2KIN cells in PBS. SK-LU-1 tumors were initiated by injecting 1×107 cells in PBS admixed with 1:1 Matrigel into flanks. Tumor volume was calculated as: 0.5 × (L × W2), where L is length and W is width. Intratumoral or peritumoral injections, as well as intraperitoneal injections of antibodies were done on multiple times at 3-4 days interval starting from day 7 for OVACR3, A427, HCT116, Brpkp110 and KPMSH2KIN tumors or from day 20 for SK- LU-1 tumors after the tumor challenge at a dose of 100μg/20g. MRTX1133 (MedChemExpress, #HY-134813) was injected daily or multiple times at 3-4 days interval starting from day 7 for OVACR3 and A427 or from day 20 for SK-LU-1 tumors at a dose of 200µg/20g. CD8 T cell depletion has been performed using αCD8 antibodies (BioXCell), injected at same frequency of injected antibodies, at a dose of 200μg/20g. Recombinant antibody production Anti-KRASG12D, and anti-IDH1R132H antibody variable heavy (VH) and variable light (VL) sequences were obtained from the patent US10844136B2 and CN106957367A, respectively. VH sequences followed by human IgA1 or IgG4 heavy chain constant region sequences in frame were cloned into PBMN-I-GFP vector (RRID:Addgene_1736). VL sequences along with human kappa light chain constant region sequences in frame were cloned into pVITRO1 (Genscript). Vectors, coding for all the VH and VL, were produced by Genscript. J-chain encoding pcDNA3.0 vector was procured from Addgene (RRID:Addgene_145146). For recombinant dimeric and monomeric KRASG12D-IgA1 and KRASG12D-IgG4 antibody production, HEK293T cells were retrovirally transduced with antibody heavy chain encoding PBMN-I-GFP vector and sorted for viable GFP+ cells, followed by transfection with antibody light chain encoding pVitro1 vector and selected using Hygromycin, and finally those cells were transfected with J-chain encoding pcDNA3.0 vector and selected using G418. Heavy chain plus light chain plus J-chain expressing HEK293T cells were grown in suspension for 5-7 days in Free-Style 293 expression Attorney Docket Number: 10110-425WO1 media (Thermo) for dimeric KRASG12D-IgA antibody production. Similarly, for monomeric KRASG12D-IgA1 or KRASG12D-IgG4, HEK293T cells expressing only light chain and respective heavy chain were used. Supernatants were concentrated through a 100 kDa membrane, to eliminate any possible contaminants of unassembled J-chains, heavy chains or light chains, and antibodies were purified using IgA or IgG purification columns (Ligatrap). Dimerization of IgA antibodies were confirmed by detecting J-chain in the elutes through mass spectrometry. All transfections were performed using Lipofectamine 3000 (Thermo). Cell lines were routinely tested for negative mycoplasma contamination. Dimeric and monomeric IDH1R132H-IgA1, and monomeric IDH1R132H-IgG4 antibodies and some additional quantity KRASG12D-IgG4 antibodies were procured from GenScript. Fluorescence-activated cell sorting (FACS) Sorting of antibody heavy chain-transduced HEK293T cells (GFP expressing) were performed by staining with DAPI (Thermo Scientific) viability dye and gated for DAPI-GFP+ cells. Similarly, PIGR-transduced HCT116 cells were sorted by gating DAPI-GFP+ cells. Samples were subsequently fluorescence-activated cell sorting (FACS) sorted using BD FACS ARIA. One hundred thousand KRASG12D transduced OVCAR3 cells were placed in 6-well plates. After 12 h wells were washed, and fresh medium was added, and cells were treated with anti-human KRASG12D-IgA1 or KRASG12D-IgG4 or non-specific human IgA (Abcam, ab91025) at 0.5 μg/ml final concentration or vehicle (PBS) and incubated for 16 h in a 37-°C incubator. Total apoptotic cells (annexin V+ propidium iodide+/−) were analyzed by flow cytometry. MTT assay Five thousand OVCAR3 (wild-type or G12D mutated KRAS-transduced or untraduced) or SK-LU-1 cells, or five hundred HEK293T cells were seeded in each well of 96-well plates. After 12 h cells were washed with PBS, and fresh medium was added, and were treated with anti-human KRASG12D-IgA1 or non-specific human IgA (Abcam, ab91025) at 0.5 μg/ml final concentration or vehicle (PBS) and the medium was changed after 16 h incubation in a 37-°C incubator to remove the treatment. Proliferation was measure at 3, 5 or 7 days. MTT reagent solution (10ul of 5mg/ml) was added to the media of cultured cells and incubated for 3 hr. The precipitates were dissolved in DMSO, after remove the medium, and absorbance were measured Attorney Docket Number: 10110-425WO1 at 570nm. Fold changes were calculated by dividing with the mean absorbance of the replicates of respective vehicle-treated cell lines. Immunofluorescence and confocal microscopy Anti-human KRASG12D-IgA1 or KRASG12D-IgG4 antibodies were conjugated with Alexa Fluor 488-conjugation kit (Thermo). Fifty thousand OVCAR3 (transduced with wild-type or G12D mutated KRAS, fused with PAmCherry) cells were placed onto a coverslip within 6-well plates and after 12 h treated with the conjugated antibodies for different temporal points. PAmCherry was photoactivated using the Leica TCS SP8 DAPI bandpass filter cube (Excitation BP 450-490 nm) using the widest aperture. Exposure time was dependent on the day of the experiment and varied between 20 to 30 seconds and was optimized using positive and negative controls. The same exposure time was used for all treatments. The gain for the Hoechst channel was adjusted to compensate for photobleaching due the exposure time required for photoactivating PAmCherry. Images were acquired with a 40X/1.3 NA objective in a confocal microscope (Leica SP8) using LAS X (v.3.5.5.19976) software. Hoechst, Alexa Fluor 488, PAmCherry were excited with laser lines 405 nm, 488 nm, 552 nm respectively. Emission was captured through band pass settings 415–480 nm, 500–525 nm, 592–651 nm, respectively. For the highest resolution images, we used the same parameters but we increased the pixel resolution to 2048 x 2048 and took the images with speed=400 and z=0.5um. Exocytosis of KRASG12D and LC-MS/MS Five hundred thousand OVCAR3 (transduced with wild-type or G12D mutated KRAS, fused with PAmCherry) cells were placed in 100mm × 20mm cell culture plates and treated with or without anti-human KRASG12D-IgA1 or KRASG12D-IgG4 or non-specific human IgA (Abcam, ab91025) at 0.5 μg/ml final concentration or vehicle (PBS) in low serum media. After 12 h, conditioned medium was collected and filtered for contaminant debris removal. Proteins were extracted from the conditioned medium, reduced by DTT, digested by trypsin, and subjected to mass spectrometry analysis by the Moffitt Cancer Center Proteomics Facility. MaxQuant (version 1.5.2.8) was used to analyze the data, identify, and quantify the proteins. Western blot, Native gel electrophoresis and Co-immunoprecipitation (Co-IP) Cells were lysed in RIPA buffer (Thermo) with protease-phosphatase inhibitor cocktail (CST, #5872S) and cleared by centrifugation. Proteins were quantified by Attorney Docket Number: 10110-425WO1 BCA assay (Thermo). Membranes were blotted with recombinantly produced anti- KRASG12D-IgA1 or anti-KRASG12D-IgG4 or anti-IDH1R132H-IgA1 or anti-IDH1R132H-IgG4 antibodies; or commercial anti-mCherry Rockland, #600-401-P16, RRID:AB_2614470), anti-human immunoglobulin heavy alpha chain (Thermo Fisher Scientific, #A18781, RRID:AB_2535558), or anti-human immunoglobulin heavy gamma (plus light) chain (Thermo Fisher Scientific, #A18805, RRID:AB_2535582), or anti-human immunoglobulin kappa chain (R&D Systems, #MAB10050), or anti- human J-chain (Thermo Fisher Scientific, Mc19-9, #MA1-80527, RRID:AB_934333), or anti-human Rab5A (Proteintech, #11947-1-AP, RRID:AB_2269388), or anti-human Rab7A (Proteintech, #55469-1-AP, RRID:AB_11182831), or anti-human Rab8A (Proteintech, #55296-1-AP, RRID:AB_10858398), or anti-human Rab11A (Proteintech, #20229-1-AP, RRID:AB_10666202), or anti-human PIGR (Abcam, #ab96196, RRID:AB_10677612), or anti-human KRASG12D (CST, #14429, D8H7, RRID:AB_2728748) or anti-human β-actin (CST, #5125, 13E5, RRID:AB_1903890), antibodies. Lysates of A427 cells treated with KRASG12D-specific IgA or MRTX1133 for different time points were probed with antibodies against total ERK1/2 (CST, #9102, RRID:AB_330744) and phosphorylated ERK1/2 (CST, D13.14.4E, #9102, RRID:AB_330744). Blots were then probed with appropriate horse radish peroxidase (HRP)-conjugated secondary antibodies (room temperature ,1-2 hour), unless the primary antibodies are HRP-conjugated, and immunoreactive bands were developed using Enhanced Chemiluminescence (ECL) substrate (GE HealthCare). For the native gel electrophoresis, 5µg of dimeric KRASG12D-IgA1, dimeric IDHR132H-IgA1, monomeric IDHR132H-IgA1 and irrIgA were diluted using Tris-Glycine Native Sample Buffer (ThermoFisher, #LC2673) and ran in a native gel, 12% Tris- Glycine Gel (Invitrogen, # XP00120BOX) for 1 hour at 220V. The gel was stained by overnight shaking with Invitrogen™ Colloidal Blue Staining Kit (Invitrogen, #LC6025). Then, the staining solution was replaced with deionized water, followed by overnight shaking until the gel had a clear background. The gel was scanned in a Canon TR4700 series HTTP. For co-immunoprecipitation, cells were lysed using non-reducing non- denaturing lysis reagent provided in the co-immunoprecipitation (co-IP) kit (Pierce, Cat#26149) used. Proteins were immunoprecipitated using anti-human PIGR- secretory component (Abcam, SC-05, #ab3924, RRID:AB_2261963) or anti-human Rab5A, or anti-human Rab7A, or anti-human Rab8A, or anti-human Rab11A, or anti- Attorney Docket Number: 10110-425WO1 human IgA (Abcam, EPR5367-76, #ab124716, RRID:AB_10976507). Proteins from concentrated conditioned media from anti-human KRASG12D-IgA1 or KRASG12D-IgG4 or non-specific human IgA or vehicle treated OVCAR3 cells were immunoprecipitated using anti-human PIGR-secretory component. The elutes were ran for western blotting. For analyzing changes in intracellular KRASG12D protein level upon antibodies-treatment, lysates of KRASG12D-transduced OVCAR3 or A427 cells, treated every 4 hours for a total of 12 hours, were used for western blotting. Transwell and transcytosis assay for expelling of KRASG12D KRASG12D OVCAR3 cells (2×105) were seeded in the upper chamber of a transwell system (CORNING® 3413 TRANSWELL® 6.5mm Polycarbonate, membrane Inserts Pre-Loaded in 24-Well Culture Plates, Pore Size: 0.4µm) in 200uL. In addition, 1 mL of fresh medium was added in the basal chamber. The cells were cultured in the incubator at 37oC and 5% of CO2 during 3 days to create a monolayer. KRASG12D IgA1 or KRASG12D IgG4 or non-specific human IgA antibodies were biotinylated using a Biotinylation Kit (Fast, Type B) by following the manufacture’s protocol. Then, 1µg of respective biotinylated antibody was added to the upper chamber. After overnight incubation, the supernatant from the upper and basal chambers were collected into separate microcentrifuge tubes and subjected to streptavidin pull-down using Dynabeads MyOne streptavidin T1. Briefly, 10µl of beads per sample were washed according to the manufacture’s recommendation, added to each sample and then incubated overnight at 4oC with head-over rotation. The beads were collected my magnetic separation for 5 min and washed 3 times in PBS containing 0.1% BSA. Finally, the beads were resuspended in 100uL of PBS and used for performing dot blots. For dot blots, 10µL of beads were blotted onto a nitrocellulose membrane and dried at room temperature 1-2 hours. Then, membranes were blocked using 5% BSA in TBS-T with a gentle agitation for 1 hour at room temperature. Appropriate primary antibodies (αhuman-IgA-HRP, αhuman-IgG-HRP and αKRASG12D) were added immediately and incubated 1 hour at room temperature with gentle rotation. After 3 washes, blots were directly chemiluminescent-developed when probed with HRP conjugated antibodies αhuman-IgA/IgG antibodies, or first incubated with a secondary antibody and developed. Multiplex immunohistochemistry Attorney Docket Number: 10110-425WO1 FFPE tissue microarray was immunostained using the PerkinElmer OPAL TM Automation IHC kit (Waltham, MA) on the BOND RX autostainer (Leica Biosystems, Vista, CA) and the following anti-human antibodies: IgA (Abcam, EPR5367-76, ab124716, RRID:AB_10976507; 1:1000), IgG (Abcam, EPR4421, ab109489, RRID:AB_10863040; 1:500), PIGR (Abcam, ab96196, RRID:AB_10677612; 1:100), CD3 (Thermo Fisher Scientific; Cat# MA5-14524, Clone SP7; RRID: AB_10982026, 1:100), and pan-cytokeratin (PCK, Dako, AE1/AE3, M3515, 1:200). Nuclei were stained with DAPI. Precisely, tissues were baked at 65ºC for 2 hours then transferred to the BOND RX (Leica Biosystems) followed by automated deparaffinization, antigen retrieval using OPAL IHC procedure (PerkinElmer). Autofluorescence slides (negative control) were included, which use primary and secondary antibodies omitting the OPAL fluors. Slides were scanned and imaged with the PerkinElmer Vectra®3 Automated Quantitative Pathology Imaging System. Multi-layer TIFF images were exported from InForm (PerkinElmer) and loaded into HALO (Indica Labs, New Mexico) for quantitative image analysis. Each fluorescent fluorophore is assigned to a dye color and positivity thresholds were determined per marker based on published nuclear or cytoplasmic staining patterns. Tumor islets and stroma were distinguished by PCK staining. 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Claims

Attorney Docket Number: 10110-425WO1 WHAT IS CLAIMED IS: 1. A bispecific antibody comprising an oncogene-binding domain and an α-PIGR binding domain; wherein the α-PIGR binding domain comprises an α-PIGR-binding variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and an α-PIGR-binding variable light (VL) domain having CDR1, CDR2 and CDR3 sequences; wherein the CDR1 sequence of the α-PIGR-binding VH domain comprises the amino acid sequence GYTFIEYT (SEQ ID NO:1); the CDR2 sequence of the α-PIGR- binding VH domain comprises the amino acid sequence INPNNGGT (SEQ ID NO:2); the CDR3 sequence of the α-PIGR-binding VH domain comprises the amino acid sequence ARYYRYDVLSAMDY (SEQ ID NO:3); the CDR1 sequence of the α-PIGR-binding VL comprises the amino acid sequence ESVDNYAISF (SEQ ID NO:4); the CDR2 sequence of the α-PIGR-binding VL domain comprises the amino acid sequence AAS; and the CDR3 sequence of the α-PIGR-binding VL domain comprises the amino acid sequence QQSKAVPYT (SEQ ID NO:5). 2. The bispecific antibody of claim 1, wherein the α-PIGR-binding VH domain comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFIEYTMHWVRQAPGQGLEWMGWINPNNG GTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARYYRYDVLSAMDYWGQ GTLVTVSS (SEQ ID NO:6). 3. The bispecific antibody of claim 1 or 2, wherein the α-PIGR-binding VL domain comprises the amino acid sequence EIVLTQSPATLSLSPGERATLSCRASESVDNYAISFLAWYQQKPGQAPRLLIYAASNRAT GIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQSKAVPYTFGQGTKLEIK (SEQ ID NO:7). 4. The bispecific antibody of any one of claims 1 to 3, wherein the oncogene is α- KRASG12D. 5. The bispecific antibody of claim 4, wherein the oncogene-binding domain comprises a oncogene-binding variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a oncogene-binding variable light (VL) domain having CDR1, CDR2 and CDR3 sequences; Attorney Docket Number: 10110-425WO1 wherein the CDR1 sequence of the oncogene-binding VH domain comprises the amino acid sequence GTFSSYA (SEQ ID NO:8); the CDR2 sequence of the oncogene- binding VH domain comprises the amino acid sequence ISRSGHST (SEQ ID NO:9); the CDR3 sequence of the oncogene-binding VH domain comprises the amino acid sequence AKRFGSIVFDY (SEQ ID NO:10); the CDR1 sequence of the oncogene- binding VL comprises the amino acid sequence QSLFNSRTRKNY (SEQ ID NO:11); the CDR2 sequence of the oncogene-binding VL domain comprises the amino acid sequence WAS; and the CDR3 sequence of the oncogene-binding VL domain comprises the amino acid sequence KQSYYHMYT (SEQ ID NO:12). 6. The bispecific antibody of claim 5, wherein the α oncogene-binding VH domain comprises the amino acid sequence MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGTFSSYAMSWVRQA PGKGLEWVSTISRSGHSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKR FGSIVFDYWGQGTLVTVSS (SEQ ID NO:13). 7. The bispecific antibody of claim 5 or 6, wherein the oncogene-binding VL domain comprises the amino acid sequence MGWSCIILFLVATATGVHSDLVMTQSPATLSLSPGERATLSCKSSQSLFNSRTRKNYLAW YQQKPGQAPRLLIYWASTRESGIPGRFSGSGSGTDFLTISSLEPEDFAVYYCKQSYYHM YTFGQGTKVEIK (SEQ ID NO:14). 8. The bispecific antibody of any one of claims 1 to 7, comprising the following formula: VLO – VHO – VLP – VHP, VLP – VHP – VLO – VHO, VHP – VLP – VHO – VLO, VHO – VLO – VHP – VLP, VLO – VHO – VHP – VLP, or VLP – VHP – VHO – VLO, wherein “VHO” is an oncogene or gene fusion -binding VH domain; wherein “VLO” is an oncogene or gene fusion -binding VL domain; wherein “VHP” is a α-PIGR-binding VH domain; wherein “VLP” is a α-PIGR-binding VL domain; and wherein “–” consists of a peptide linker or a peptide bond.
Figure imgf000062_0001
Attorney Docket Number: 10110-425WO1 9. The bispecific antibody of any one of claims 1 to 7, comprising a first polypeptide, second polypeptide, third polypeptide, and fourth polypeptide, wherein the first polypeptide and second polypeptide have the formula: VHO – CH1 = CH2 – CH3 – VLP – VHP, or VHO – CH1 = CH2 – CH3 – VHP – VLP; wherein the third polypeptide and fourth polypeptide have the formula VLO – CL; wherein “CH1” is a first heavy chain constant domain; wherein “CH1” is a second heavy chain constant domain; wherein “CH1” is a third heavy chain constant domain; wherein “CL” is a light chain constant domain; wherein “VHO” is an oncogene or gene fusion-binding VH domain; wherein “VLO” is an oncogene or gene fusion-binding VL domain; wherein “VHP” is a α-PIGR-binding VH domain; wherein “VLP” is a α-PIGR-binding VL domain; wherein “–” consists of a peptide linker or a peptide bond; wherein “=” is a heavy chain hinge domain; and wherein the first polypeptide and second polypeptide are conjugated to each other at their hinge domains, wherein the third polypeptide is conjugated to the first polypeptide at their CL and CH1 domains, respectively, and wherein the fourth polypeptide is conjugated to the second polypeptide at their CL and CH1 domains, respectively. 10. The bispecific antibody of any one of claims 1 to 9, wherein the oncogene is selected from the group consisting of ABL1, ABL2, AKT1, AKT2, ALK, APC, AR, ARID1A, ARID2, ATM, BCLAF1, BRAF, BRCA1, BRCA2, CCND3, CTNNB1, CREBBP, DNMT3A, EGFR, EP300, ERBB2, ERBB3, ESR1, EZH2, FAT1, FAT3, FAT4, FBXO11, FBXW7, FGFR1, FLT3, FOXA1, GNA11, GNAQ, GNAS, GTF2I, H3F3A, HER2/NEU, HRAS, IDH1, IDH2, JAK2, KCNJ4, KDM6A, KIT, KMT2C, KMT2D, KRAS, LRP1B, MET, MTOR, MYC, MYCN, NF1, NOTCH1, NRAS, NSD1, NSD2, NTRK3, OBSCN, PCBP1, PIK3CA, PIK3R1, PIGR, PLCG1, PTCH1, PTEN, PTPN6, PTPN11, PTPN13, RAC1, RB1, RET, ROS1, RHOA, RYR2, SET2D, SF3B1, SMAD2, SMAD4, SMARCA4, SRC, SRSF2, TP53, TRRAP, TTN, U2AF1L4, VHL, and CDKN2A. Attorney Docket Number: 10110-425WO1 11. The bispecific antibody of any one of claims 1 to 9, wherein the fusion is selected from the group consisting of EML4-ALK, KIF5B-ALK, HIP1-ALK, KLC1-ALK, DCTN1- ALK, PRKAR1A-ALK, STRN-ALK, CLTC-ALK, MPRIP-ALK, NPM1-ALK, TNS1-ALK, ACTG2-ALK, IGFBP5-ALK, SEC31A-ALK, TPM3-ALK, AITC-ALK, TPM4-ALK, CD74 ROS1, SLC34A2-ROS1, SDC4-ROS1, EZR-ROS1, LR1G3-ROS1, SDC4-ROS1, TPM3- ROS1, LIMA1-ROS1, MSN-ROS1, WNK1-ROS1, RBPMS-ROS1; KIF5B-RET, CCDC6- RET, and TRIM33-RET. 12. A method of treating a cancer in a subject comprising administering to the subject the bispecific antibody of any of claims 1 to 11.
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