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EP-4739396-A1 - HUMANIZED 40H3 ANTIBODY

EP4739396A1EP 4739396 A1EP4739396 A1EP 4739396A1EP-4739396-A1

Abstract

Disclosed are antibodies ( e.g ., humanized monoclonal antibodies and antigen binding fragments) that specifically bind epidermal growth factor receptor variant III (EGFRvIII) and/or gene-amplified EGFR, as well as conjugates and chimeric antigen receptors (CARs) including such antibodies. Also provided are nucleic acid molecules encoding an antibody, conjugate, or CAR disclosed herein, and host cells expressing the nucleic acid molecules. Further disclosed are methods of using the disclosed compositions, for example, for treating or detecting a tumor, such as a tumor expressing EGFRvIII and/or gene-amplified EGFR.

Inventors

  • FITZGERALD, DAVID J.
  • ANTIGNANI, ANTONELLA
  • SARNOVSKY, ROBERT

Assignees

  • The United States of America, as represented by The Secretary, Department of Health and Human Services

Dates

Publication Date
20260513
Application Date
20240708

Claims (20)

  1. 1. An isolated humanized monoclonal antibody or antigen binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL) comprising: a) SEQ ID NO: 5 and SEQ ID NO: 8, respectively (A10, VH3+VL3); b) SEQ ID NO: 3 and SEQ ID NO: 6, respectively (A2, VH1+VL1); c) SEQ ID NO: 3 and SEQ ID NO: 7, respectively (A3, VH1+VL2); d) SEQ ID NO: 3 and SEQ ID NO: 8, respectively (A4, VH1+VL3); e) SEQ ID NO: 4 and SEQ ID NO: 6, respectively (A5, VH2+VL1); f) SEQ ID NO: 4 and SEQ ID NO: 7, respectively (A6, VH2+VL2); g) SEQ ID NO: 4 and SEQ ID NO: 8, respectively (A7, VH2+VL3); h) SEQ ID NO: 5 and SEQ ID NO: 6, respectively (A8, VH3+VL1); i) SEQ ID NO: 5 and SEQ ID NO: 7, respectively (A9, VH3+VL2); j) SEQ ID NO: 1 and SEQ ID NO: 9, respectively (Bl, 40H3 VH+VL-EG); k) SEQ ID NO: 1 and SEQ ID NO: 10, respectively (B2, 40H3 VH+VL-DA); l) SEQ ID NO: 4 and SEQ ID NO: 11, respectively (B3, VH2+VL1-DA); m) SEQ ID NO: 4 and SEQ ID NO: 12, respectively (B4, VH2+VL2-DA); n) SEQ ID NO: 5 and SEQ ID NO: 11, respectively (B5, VH3 and VL1-DA); o) SEQ ID NO: 31 and SEQ ID NO: 32, respectively (CIO); p) SEQ ID NO: 5 and SEQ ID NO: 14, respectively (D2); or q) SEQ ID NO: 5 and SEQ ID NO: 24, respectively (D3).
  2. 2. The isolated humanized monoclonal antibody of claim 1, wherein the antibody comprises a human constant domain.
  3. 3. The isolated humanized monoclonal antibody of claim 1 or claim 2, wherein the antibody is an IgG.
  4. 4. The isolated humanized monoclonal antibody of any one of the prior claims, comprising a recombinant constant domain comprising a modification that increases the half-life of the antibody.
  5. 5. The isolated humanized monoclonal antibody or antigen binding fragment of any one of the prior claims, conjugated to a toxin or a chemotherapeutic agent.
  6. 6. The isolated humanized monoclonal antibody or antigen binding fragment of claim 5, wherein the toxin is a Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin, ribotoxin, ribonuclease, saporin, calicheamicin, or a botulinum toxin.
  7. 7. The isolated humanized monoclonal antibody or antigen binding fragment of claim 5 or claim 6, wherein the toxin is the PE, and wherein the PE is PE25, PE38 or PE40.
  8. 8. The isolated humanized monoclonal antibody or antigen binding fragment of a claim 5, wherein the chemotherapeutic agent is monomethyl auristatin E or a maytansinoid.
  9. 9. The antigen binding fragment of any one of the prior claims.
  10. 10. The antigen binding fragment of any one of the prior claims, wherein the antigen binding fragment is a Fv, dsFV, ds-scvFV, Fab, F(ab')2, scFV or a scFVi fragment.
  11. 11. The antigen binding fragment of any one of the prior claims, wherein the antigen binding fragment is a Fab or scFV.
  12. 12. The isolated humanized monoclonal antibody or antigen binding fragment of any one of the prior claims, conjugated to a detectable marker.
  13. 13. A chimeric antigen receptor (CAR) comprising the antigen binding fragment of any one of the prior claims.
  14. 14. A bispecific antibody comprising the humanized monoclonal antibody or antigen binding fragment of any one of claims 1-11.
  15. 15. An isolated nucleic acid molecule encoding the humanized antibody or antigen binding fragment of any one of claims 1-12, or a VH or VL of the antibody or antigen binding fragment, the CAR of claim 13, or the bispecific antibody of claim 14.
  16. 16. The isolated nucleic acid molecule of claim 15, wherein the nucleic acid molecule is a cDNA sequence.
  17. 17. The isolated nucleic acid molecule of claim 15 or claim 16, operably linked to a promoter.
  18. 18. A vector comprising the nucleic acid molecule of any one of claims 15-17.
  19. 19. The vector of claim 18, wherein the vector is a viral vector.
  20. 20. An isolated host cell comprising the nucleic acid molecule of any one of claims 15-17, or the vector of claim 18 or 19.

Description

HUMANIZED 40H3 ANTIBODY CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63/525,493, filed July 7, 2023, which is incorporated by reference in its entirety. FIELD OF THE DISCLOSURE This application is related to the field of cancer biology, specifically to humanized monoclonal antibodies and antigen binding fragments that specifically bind epidermal growth factor receptor mutants/variants expressed by tumor cells (e.g., EGFRvIII and/or gene-amplified EGFR). STATEMENT OF GOVERNMENT SUPPORT This invention was made with Government support under project number Z01#: Z1 A BC 008757 by the National Institutes of Health, National Cancer Institute. The United States Government has certain rights in the invention. SEQUENCE LISTING REFERENCE The Sequence Listing is submitted as an XML file named “Sequence. xml,” created on July 8, 2024, (1,386,226 bytes), which is incorporated herein by reference. BACKGROUND EGFR is frequently involved in the oncogenic progression of human cancer. Various alterations in expression including gene amplifications and activating mutations contribute to oncogenesis. The human form of this large receptor has an external domain (ECD) of 621 amino acids, a single pass transmembrane domain (TM) of 23 amino acids and an enzymatically active intracellular domain (ICD) of 542 amino acids. EGFR is a member of the receptor tyrosine kinase family and was the first receptor shown to be associated positively with human cancer. Ligand binding leads to receptor dimer formation and the activation of the kinase domain which signals to one of several pathways that can promote the growth, survival and spread of mammalian cells. Activating mutations can occur in either the ECD or the ICD. Gene amplifications and deletions can also activate EGFR. Exemplary deletions include the loss of exons 2-7, which produces EGFR variant III (EGFRvIII), and the loss of exon 19 generates a constitutively active enzyme mutant. Gene amplification of EGFR, expression of EGFRvIII, or the loss of exon 19 of EGFR are reported only in cancer cells. Antibodies generally bind their target antigen tightly and specifically, making them usefill therapeutics for treating a wide range of diseases characterized by altered protein expression, such as the expression of EGFRvIII or gene-amplified EGFR. For many therapeutic applications, however, the efficacy and safety of non-human antibodies is compromised because non-human immunoglobulin (Ig) molecules are themselves immunogenic, and thus are capable of inducing an anti-Ig immune response, making repeat dosing highly compromised. Thus, for therapeutic use, it can be beneficial to modify an antibody to reduce or eliminate immunogenicity. A need remains for humanized antibodies that can bind EGFRvIII or gene-amplified EGFR, but not wild-type EGFR, to exhibit cancer cell specificity. SUMMARY OF THE DISCLOSURE Disclosed are isolated humanized monoclonal antibodies and antigen binding fragments thereof, wherein the monoclonal antibody specifically binds to EGFRvIII and/or gene-amplified EGFR. In some aspects, the monoclonal antibody or antigen binding fragment includes a heavy chain variable region (VH) and a light chain variable region (Vj comprising: a) SEQ ID NO: 5 and SEQ ID NO: 8, respectively (A10, VH3+VL3); b) SEQ ID NO: 3 and SEQ ID NO: 6, respectively (A2, VH1+VL1); c) SEQ ID NO: 3 and SEQ ID NO: 7, respectively (A3, VH1+VL2); d) SEQ ID NO: 3 and SEQ ID NO: 8, respectively (A4, VH1+VL3); e) SEQ ID NO: 4 and SEQ ID NO: 6, respectively (A5, VH2+VL1); f) SEQ ID NO: 4 and SEQ ID NO: 7, respectively (A6, VH2+VL2); g) SEQ ID NO: 4 and SEQ ID NO: 8, respectively (A7, VH2+VL3); h) SEQ ID NO: 5 and SEQ ID NO: 6, respectively (A8, VH3+VL1); i) SEQ ID NO: 5 and SEQ ID NO: 7, respectively (A9, VH3+VL2); j) SEQ ID NO: 1 and SEQ ID NO: 9, respectively (Bl, 40H3 VH+VL-EG); k) SEQ ID NO: 1 and SEQ ID NO: 10, respectively (B2, 40H3 VH+VL-DA); l) SEQ ID NO: 4 and SEQ ID NO: 11, respectively (B3, VH2+VL1-DA); m) SEQ ID NO: 4 and SEQ ID NO: 12, respectively (B4, VH2+VL2-DA); n) SEQ ID NO: 5 and SEQ ID NO: 11, respectively (B5, VH3 and VL1-DA); o) SEQ ID NO: 31 and SEQ ID NO: 32, respectively (CIO); p) SEQ ID NO: 5 and SEQ ID NO: 14, respectively (D2); or q) SEQ ID NO: 5 and SEQ ID NO: 24, respectively (D3). Conjugates of these antibodies and antigen binding fragments are also disclosed. Further disclosed are chimeric antigen receptors (CARs) including a disclosed antibody or antigen binding fragment, and cells (e.g., immune cells) expressing such chimeric antigen receptors (e.g., CAR T cells). Also disclosed are nucleic acid molecules encoding a VH and/or a VL of any of the antibodies disclosed herein, vectors including these nucleic acids, and host cells transformed with these nucleic acids and/or vectors. In further aspects, disclosed is the use of a monoclonal antibody or antigen binding fragment disclosed herein for inhibiting a tumor that