IP Library › Granted Patent US 12,492,490
Granted Patent B2
US 12,492,490 · App. 17/880,556 · Granted Dec 9, 2025

Tumor selective macropinocytosis-dependent rapidly internalizing antibodies

Inventor: Bin Liu (San Francisco, CA)
Assignee: The Regents of the University of California
C40B30/06A61K45/06A61K47/6801A61K47/6851A61K47/6889C07K16/005C07K16/2866C07K16/30C07K16/3069C12N15/1037G01N33/574G01N33/6854C07K2317/10C07K2317/21C07K2317/32C07K2317/622C07K2317/73C07K2317/77
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Quick Facts
Patent No.
US 12,492,490
App. No.
17/880,556
Granted
Dec 9, 2025
Kind
B2
Abstract

Methods are provided for identifying and selecting antibodies that are internalized into cells via the macropinocytosis pathway. Additionally antibodies that are internalized via this pathway are provided as well as immunoconjugates comprising such antibodies.

Claims (28)

1 . A method of preparing antibodies that are internalized into a cell by a macropinocytosis pathway, said method comprising:

contacting target cells with members of an antibody library and with a marker for macropinocytosis;

identifying one or more internalized antibodies that co-localize in said target cells with said marker for macropinocytosis; and

quantifying the colocalization between said one or more internalized antibodies that co-localize and said marker for macropinocytosis, wherein the quantification comprises measuring a Pearson's correlation coefficient (PCC) value of the colocalization between said one or more internalized antibodies and said marker for macropinocytosis.

2 . The method of claim 1 , wherein:

said members of an antibody library are members of a phage display library or a yeast display library; and/or

said antibody library is an antibody library that is enriched for antibodies that bind to tumor cells; and/or

said antibody library is an antibody library that is enriched for antibodies that bind to tumor cells and said enrichment is by laser capture microdissection (LCM) of antibodies that bind to tumor cells; and/or

said antibody library is an antibody library that is enriched for antibodies that are internalized into tumor cells.

3 . The method of claim 1 , wherein said marker for macropinocytosis comprises a marker selected from the group consisting of high molecular weight dextran, latex beads, glass beads, Lucifer yellow, and soluble enzymes such as horseradish peroxidase.

4 . The method of claim 1 , wherein said contacting comprises incubating said members of an antibody library and/or said marker for macropinocytosis with said cells.

5 . The method according to claim 1 , wherein the contacting comprises incubating the members of an antibody library and/or the marker for macropinocytosis with the target cells for a period of at least 1 hour, or at least 2 hours, or at least 3 hours, or at least 4 hours, or at least 6 hours, or at least 8 hours, or at least 10 hours, or at least 12 hours, or at least 16 hours, or at least 20 hours, or at least 24 hours.

6 . The method of claim 1 , wherein the one or more internalized antibodies colocalizes with a lysosomal marker.

7 . The method of claim 5 , wherein:

said selecting comprises recovering the antibody from the sample used in the HCS analysis; and/or

selecting the antibodies from the library corresponding to the antibodies identified in the HCS analysis; and/or

said selecting comprises determining the amino acid sequence of said antibody; and/or

said selecting comprises converting said antibody into an intact immunoglobulin.

8 . The method of claim 6 , wherein the lysosomal marker is LAMP1.

9 . The method of claim 1 , further comprising selecting one or more internalized antibodies having a measured PCC value greater than 2-fold compared to the PCC value of a control antibody.

10 . The method according claim 1 , wherein the marker for macropinocytosis is labeled with a detectable label.

11 . The method of claim 10 , wherein the marker for macropinocytosis comprises labeled high molecular weight dextran.

12 . The method of claim 3 , wherein, wherein the marker for macropinocytosis comprises latex beads or glass beads.

13 . The method according to claim 1 , wherein the target cells comprise cells of one or more tumor cell lines.

14 . The method of claim 13 , wherein the one or more tumor cell lines are selected from the group consisting of PC3, DU145, HeLa, MDA-MB-231, Hs5786, MDA-435, BT549, SKOV3, HeyA8, OVCAR3, PANC1, MIAPaCa2, BxPC3, T24, TCCSUP, UMUC-3, TEI, AGS, SGC-7901, M28, VAMT-1, A549, A431, A172MG, DBTRG-5MG, U-251MG, U87MG, T84, THP1, U373, U937, VCaP, SiHa, FM3, DuCaP, A253, A172, 721, SiHa, and LNCaP.

15 . The method according to claim 1 , wherein the identifying comprises high content screening (HCS) of said cells.

16 . The method according to claim 1 , wherein the one or more colocalized antibodies is labeled with a fluorescent label attached to a second antibody that binds said colocalized antibody.

17 . The method of claim 16 , wherein the second antibody comprises an anti-fd bacteriophage.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 19, 2023
From: LIU, BIN
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 063381/0941 →
Continuity (4)
Division 16714566 · Dec 13, 2019
Division 15321684
Provisional Application 62023689 · Jul 11, 2014
Related Publication 20230227574A1 · Jul 20, 2023
References Cited (44)
US 10550195B2 · Liu · 2020 [cited by applicant]
US 11447563B2 · Liu · 2022 [cited by applicant]
US 20130065227A1 · Bates et al. · 2013 [cited by applicant]
US 20170137532A1 · Liu · 2017 [cited by applicant]
US 20200216556A1 · Liu · 2020 [cited by applicant]
WO WO9822149A1 · 1998 [cited by applicant]
WO WO2007030642A2 · 2007 [cited by applicant]
WO WO2012012759A2 · 2012 [cited by applicant]
WO WO2013109580A1 · 2013 [cited by applicant]
AU Office Action dated Mar. 20, 2020 issued in AU 2015287749. [cited by applicant]
CA Office Action dated Jul. 5, 2021, in application No. CA2954041. [cited by applicant]
CA Office Action dated Jun. 10, 2022, in Application No. CA2954041. [cited by applicant]
Chakraborty et al. (2012) “Kaposi's sarcoma-associated herpesvirus interacts with EphrinA2 receptor to amplify signaling essential for productive infection” PNAS 109(19): E1163-E1172. [cited by applicant]
EP Extended Search Report dated Jan. 2, 2018 issued in EP 15818722.9. [cited by applicant]
European Office Action dated Dec. 20, 2018 issued in EP 15818722.9. [cited by applicant]
Ha et al. (2014) “High-content Analysis of Antibody Phage-display Library Selection Outputs Identifies Tumor Selective Macropinocytosis-dependent Rapidly Internalizing Antibodies” Mo. Cell. Proteom. 13(12):3320-3331. [cited by applicant]
Ha et al. (2016) “Macropinocytosis Exploitation by Cancers and Cancer Therapeutics” Frontiers in Physiology 7(381):1-10. [cited by applicant]
Hewlett et al. (1994) “The coated pit and macropinocytic pathways serve distinct endosome populations” J. Cell Biol. 124: 689-703. [cited by applicant]
Jackson et al. (2008) “A Human Antibody-Drug Conjugate Targeting EphA2 Inhibits Tumor Growth In vivo” Cancer Res. 68: 9367-9374. [cited by applicant]
Johns T.G., et al., “Novel Monoclonal Antibody Specific for the de2-7 Epidermal Growth Factor Receptor (EGFR) That Also Recognizes the EGFR Expressed in Cells Containing Amplification of the EGFR Gene”, International Jo… [cited by applicant]
Kubo et al. (2008) “Identification of oligopeptide binding to colon cancer cells separated from patients using laser capture microdissection” Journal of Drug Targeting 16(5): 396-404. [cited by applicant]
Liu et al. (2004) “Mapping Tumor Epitope Space by Direct Selection of Single-Chain Fv Antibody Libraries on Prostate Cancer Cells” Cancer Res. 64: 704-710. [cited by applicant]
PCT International Preliminary Report on Patentability and Written Opinion dated Jan. 26, 2017 issued in PCT/US2015/039741. [cited by applicant]
PCT International Search Report and Written Opinion dated Sep. 21, 2015 issued in PCT/US2015/039741. [cited by applicant]
Poul et al. (2000) “Selection of tumor-specific internalizing human antibodies from phage libraries” J. Mol. Biol. 301: 1149-1161. [cited by applicant]
Reyes-Reyes et al. (2010) “A New Paradigm for Aptamer Therapeutic AS1411 Action: Uptake by Macropinocytosis and Its Stimulation by a Nucleolin-Dependent Mechanism” Cancer Res. 70: 8617-8629. [cited by applicant]
Ritchie et al. (2013) “Implications of receptor-mediated endocytosis and intracellular trafficking dynamics in the development of antibody drug conjugates” Landes Bioscience 5(1): 13-21. [cited by applicant]
Ruan et al. (2006) “Identification of Clinically Significant Tumor Antigens by Selecting Phage Antibody Library on Tumor Cells in Situ Using Laser Capture Microdissection” Mol. Cell Proteomics. 5: 2364-2373. [cited by applicant]
Rudnick et al. (2011) “Influence of Affinity and Antigen Internalization on the Uptake and Penetration of Anti-HER2 Antibodies in Solid Tumors” Cancer Res. 71: 2250-2259. [cited by applicant]
Shen et al. (2013) “Enhancing Chemotherapy Response with Sustained EphA2 Silencing Using Multistage Vector Delivery” Clin. Cancer Res. 19: 1806-1815. [cited by applicant]
Sutherland et al. (2006) “Lysosomal Trafficking and Cysteine Protease Metabolism Confer Target-specific Cytotoxicity by Peptide-linked Anti-CD30-Auristatin Conjugates” J. Biol. Chem. 281: 10540-10547. [cited by applicant]
Tanaka et al. (2010) “Sustained Small Interfering RNA Delivery by Mesoporous Silicon Particles” Cancer Res. 70: 3687-3696. [cited by applicant]
Tandon et al. (2011) “Emerging strategies for EphA2 receptor targeting for cancer therapeutics.” Expert Opin. Ther. Targets. 15: 31-51 [NIH Public Access—Author Manuscript—31 pages] doi:10.1517/14728222.2011.538682. [cited by applicant]
U.S. Office Action dated Oct. 15, 2021, in U.S. Appl. No. 16/714,566. [cited by applicant]
U.S. Notice of Allowance dated May 9, 2022 in U.S. Appl. No. 16/714,566. [cited by applicant]
US Notice of Allowance dated Sep. 18, 2019 issued in U.S. Appl. No. 15/321,684. [cited by applicant]
US Office Action dated Apr. 5, 2019 issued in U.S. Appl. No. 15/321,684. [cited by applicant]
US Office Action [Restriction Requirement] dated Jan. 15, 2019 issued in U.S. Appl. No. 15/321,684. [cited by applicant]
Veithen et al. (1996) “v-Src induces constitutive macropinocytosis in rat fibroblasts.” J. Cell Sci. 109(Pt 8): 2005-2012. [cited by applicant]
Wang et al. (2014) “Macropinosome quantitation assay” MethodsX I: 36-41. [cited by applicant]
West et al. (1989) “Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells.” J. Cell Biol. 109: 2731-2739). [cited by applicant]
Wykosky and Debinski (2008) “The EphA2 Receptor and EphrinA1 Ligand in Solid Tumors: Function and Therapeutic Targeting” Mol. Cancer Res. 6: 1795-1806. [cited by applicant]
Zhou et al. (2010) “Internalizing cancer antibodies from phage libraries selected on tumor cells and yeast-displayed tumor antigens.” J. Mol. Biol. 404: 88-99 [NIH Public Access—Author Manuscript—24 pages] doi:10.1016/j… [cited by applicant]
Zhu et al. (2010) “Identification of Internalizing Human Single-Chain Antibodies Targeting Brain Tumor Sphere Cells” Mol. Cancer Ther. 9: 2131-2141. [cited by applicant]