WO2024050439A2 - Anticorps tétravalent biépitopique ciblant l'egfr - Google Patents

Anticorps tétravalent biépitopique ciblant l'egfr Download PDF

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WO2024050439A2
WO2024050439A2 PCT/US2023/073194 US2023073194W WO2024050439A2 WO 2024050439 A2 WO2024050439 A2 WO 2024050439A2 US 2023073194 W US2023073194 W US 2023073194W WO 2024050439 A2 WO2024050439 A2 WO 2024050439A2
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antibody
domain
seq
biepitopic
scfv
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PCT/US2023/073194
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WO2024050439A3 (fr
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Luke A. HELGESON
Tyler C. ROZANITIS
Dennis R. GOULET
Jahan Khalili
Nicholas S. DAVIS
Yi Zhu
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Systimmune, Inc.
Baili-Bio (Chengdu) Pharmaceutical Co., Ltd.
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Publication of WO2024050439A2 publication Critical patent/WO2024050439A2/fr
Publication of WO2024050439A3 publication Critical patent/WO2024050439A3/fr

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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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • 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
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • BIEPITOPIC TETRAVALENT ANTIBODY TARGETING EGFR CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Application Ser. No.63/402,945 filed August 31, 2022, under 35 U.S.C.119(e), the entire disclosures of which are incorporated by reference herein.
  • TECHNICAL FIELD The present disclosure generally relates to the technical field of antibody cancer therapeutics, and more particularly relates to biepitopic tetravalent antibodies.
  • BACKGROUND Glioblastoma multiforme is an aggressive form of cancer accounting for the majority of malignancies originating in the brain and has poor prognosis with an average survival of 14-15 months post diagnosis.
  • glioblastoma cases 34-63%
  • a large percentage of glioblastoma cases (34-63%) involve amplification of the EGFR gene.
  • mutant versions of EGFR occur in 63-75% of cases, the most common of which is called EGFRvIII and occurs in 25- 64% of cases.
  • EGFRvIII mutation results in a 267-residue deletion of the extracellular domain of the protein, which functionally may induce constitutive signaling due to less efficient endocytosis of the receptor. 2
  • These changes cause EGFRvIII-bearing tumors to display increased proliferation, increased angiogenesis, and reduced apoptosis.
  • wild-type EGFR and mutant EGFRvIII may also be overexpressed in other types of solid tumor, including ovarian, breast, and lung cancers.
  • EGFR Several antibodies targeting EGFR, including Cetuximab, Panitumumab, and Necitumumab, have been approved by the FDA for treatment of epithelial tumors.
  • Nimotuzumab is also approved in several countries for treatment of solid tumors. The lower affinity of Nimotuzumab for EGFR, compared to Cetuximab or Panitumumab, could help to explain its reduced skin toxicities.
  • antibody-based therapies specifically targeting mutant EGFRvIII have been evaluated in clinical trials, including the monoclonal antibody Depatuxizumab (Phase I) and the antibody-drug conjugate Depatuxizumab mafodotin (Phase 3).
  • Phase 3 trial for the EGFRvIII-targeting ADC was halted after not meeting the primary endpoint of overall survival in patients with newly diagnosed glioblastoma.
  • 4 T cell engagers targeting EGFRvIII and CD3 have also been evaluated preclinically, further highligthing the promise of EGFRvIII as a therapeutic target.
  • the disclosure provides biepitopic tetravalent antibodies targeting at least two epitopes of EGFR, and the methods of making and using the antibody.
  • the application provides a biepitopic tetravalent antibody having a binding affinity to at least two epitopes of EGFR.
  • the antibody may include an antibody backbone, comprising an antibody light chain having an antibody light chain variable (VL) domain, an antibody heavy chain having an antibody heavy chain variable (VH) domain, wherein the antibody VL domain and the antibody VH domain forms a Fab region.
  • the antibody may further include a scFv domain having a scFv light chain variable (VL) domain and scFv heavy chain variable (VH) domain, wherein the scFv domain is linked to at least one end of the antibody light chain or the antibody heavy chain through an inter-domain linker.
  • each scFv domain has a structure order of N terminus – VH – linker – VL – C terminus, or N-terminus – VL– linker– VH– C-terminus.
  • the linker comprises a flexible GS linker having from about 20 amino acids.
  • the linker comprises an amino acid sequence (Gly-Gly-Gly-Gly-Ser)m, and wherein m is an integer of at least 3. In one embodiment, m is 4. In one embodiment, the inter-domain linker comprises from about 10 to about 30 amino acids. In one embodiment, the inter-domain linker comprises an amino acid sequence (Gly-Gly- Gly-Gly-Ser)m, and wherein m is an integer of at least 2. In one embodiment, m is an integer from 2 to 6.
  • the two epitopes of EGFR comprise an EGFR wild-type (EGFRwt) epitope and an EGFRvIII epitope, and the biepitopic tetravalent antibody has a stronger binding affinity to EGFRvIII than to EGFRwt.
  • the antibody has a binding affinity to the EGFRwt epitope with a first KD, and a binding affinity to the EGFRvIII epitope with a second KD, wherein the first KD is higher than the second KD.
  • the first KD is not less than 1E-11 M
  • the second KD is not more than 1E-06 M.
  • the first KD is between 1E-10 M and 1E-06 M
  • the second KD is between 1E-11 M and 1E-07 M.
  • the first KD is more than 1.1-fold, 1.2- fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60- fold, or 70-fold higher than the second KD.
  • the first KD is between 1.1 to 1000-fold stronger than the second KD.
  • the antibody has a first ADCC EC50 against cells bearing only EGFRwt and a second ADCC EC50 against cells bearing EGFRwt and EGFRvIII, wherein the first ADCC EC50 is more than the second ADCC EC50.
  • the first ADCC EC50 is more than 1.1- fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold higher than the second ADCC EC50.
  • the EGFRwt epitope comprises the epitope having a binding affinity to Cetuximab.
  • the EGFRvIII epitope comprises the epitope having a binding affinity to ABT-806.
  • the scFv domain of the biepitopic tetravalent antibody has the binding affinity to the EGFRwt epitope and the Fab domain has the binding affinity to the EGFRvIII epitope. In one embodiment, the scFv domain has the binding affinity to the EGFRvIII epitope and the Fab domain has the binding affinity to the EGFRwt epitope. In one embodiment, the scFv domain is linked to the biepitopic tetravalent antibody light chain at its C-terminus. In one embodiment, the scFv domain is linked to the antibody light chain at its N-terminus. In one embodiment, the scFv domain is linked to the antibody heavy chain at its C-terminus.
  • the scFv domain is linked to the antibody heavy chain at its N-terminus.
  • the antibody backbone may be derived from Cetuximab, humanized Cetuximab, dematured Cetuximab, or humanized dematured Cetuximab (i.e., Cetuximab-derived backbone), or Nimotuzumab, and the scFv domain may comprise a binding domain derived from or ABT-806 or humanized ABT-806.
  • the antibody backbone may be derived from ABT-806 or humanized ABT- 806, and the scFv domain may comprise a binding domain derived from Cetuximab, humanized Cetuximab, dematured Cetuximab, or humanized dematured Cetuximab, or Nimotuzumab.
  • the humanized ABT-806 is ABT-806 V1, V2, V3, V4, V5, V6, V7, V8, or V9.
  • Cetuximab backbone biepitopic tetravalent antibodies provides a biepitopic tetravalent, wherein the antibody backbone is derived from Cetuximab, humanized Cetuximab, dematured Cetuximab, or humanized dematured Cetuximab (i.e., Cetuximab-derived backbone).
  • the scFv domain comprises a binding domain derived from the variable region of ABT-806 or humanized ABT-806.
  • the antibody backbone comprises a Cetuximab dematuration mutation (sequential numbering) selected from VH-Y101, VH-Y102, VH-D103, VH-Y104, VL-N92, or a combination thereof.
  • the Cetuximab dematuration mutation comprises VH-Y101A, VH-Y101W, VH-Y102A, VH-D103F, VH-D103W, VH- D103Y, VH-Y104L, VL-N92F, VL-N92K, or a combination thereof.
  • the antibody heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.1, 13, 79, 97, 101, 103, 105, 107, 123, 127, 129, 131, or 133.
  • the antibody light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.3, 81, 83, 99, 109, 125, or 135.
  • the antibody backbone comprises humanized Cetuximab.
  • the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.
  • the antibody VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.239.
  • the antibody backbone comprises dematured Cetuximab.
  • the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.247, 251, 253, 259, 261, 263, 265, or 267.
  • the antibody VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.243 or 269.
  • the antibody backbone comprises the humanized dematured Cetuximab.
  • the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.245, 249, 255, or 257.
  • the antibody VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.241.
  • the humanized ABT-806 is ABT-806 V1, V2, V3, V4, V5, V6, V7, V8, or V9.
  • the scFv domain comprises the variable regions of ABT-806 or humanized ABT-806.
  • the scFv VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.209, 225, 229, or 233.
  • the scFv VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.211, 227, 231, or 235.
  • the antibody VH domain comprises 3 complementary determining regions (CDRs) of SEQ ID NO.201, 237, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, or 267.
  • the antibody VL domain comprises 3 CDRs of SEQ ID NO.203, 239, 241, 243, or 269.
  • the scFv VH domain comprises 3 CDRs of SEQ ID NO. 209.
  • the scFv VL domain comprises 3 CDRs of SEQ ID NO.211.
  • the biepitopic tetravalent antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO. 1, 3, 13, 79, 81, 83, 97, 99, 101, 103, 105, 107, 109, 123, 125, 127, 129, 131, 133, or 135.
  • the application provides a biepitopic tetravalent antibody, wherein the antibody backbone is derived from ABT0806 or humanized ABT-806, and wherein the scFv domain comprises a binding domain derived from the variable region of Cetuximab, humanized Cetuximab, dematured Cetuximab, humanized dematured Cetuximab. In one embodiment, the scFv domain comprises a binding domain derived from Nimotuzumab.
  • the antibody heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.9, 17, 19, 21, 23, 25, 27, 29, 31, 75, 93, 113, 115, 117, 119, 121, 137, 139, 141, 143, 145, 147, or 179.
  • the antibody light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.11, 33, 35, 37, 39, 41, 43, 77, 91, 95, or 111.
  • the antibody backbone comprises the ABT-806, and wherein the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.209.
  • the VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.211.
  • the antibody backbone comprises the humanized ABT-806.
  • the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.225, 229, or 233.
  • the antibody VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.227, 231, or 235.
  • the scFv VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.201, 205, 237, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, or 267.
  • the scFv VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.203, 207, 239, 241, 243, or 269.
  • the scFv domain comprises a binding domain derived from the variable region of the dematured Cetuximab or the humanized dematured Cetuximab.
  • the dematured Cetuximab or the humanized dematured Cetuximab comprises a Cetuximab dematuration mutation (sequential numbering) selected from Y101, Y102, D103, Y104, N92, and a combination thereof.
  • the Cetuximab dematuration mutation comprises VH-Y101, VH-Y102, VH-D103, VH-Y104, VL-N92, or a combination thereof. In one embodiment, the Cetuximab dematuration mutation (sequential numbering) comprises VH-Y101A, VH-Y101W, VH-Y102A, VH-D103F, VH-D103W, VH-D103Y, VH- Y104L, VL-N92F, VL-N92K, or a combination thereof. In one embodiment, the biepitopic tetravalent antibody VH domain comprises 3 complementary determining regions (CDRs) of SEQ ID NO. 209.
  • CDRs complementary determining regions
  • the biepitopic tetravalent antibody VL domain comprises 3 CDRs of SEQ ID NO. 211.
  • the scFv VH domain comprises 3 CDRs of SEQ ID NO.201, 205, 237, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, or 267.
  • the scFv VL domain comprises 3 CDRs of SEQ ID NO.203, 207, 239, 241, 243, or 269.
  • the biepitopic tetravalent antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 41, 43, 75, 77, 91, 93, 95, 111, 113, 115, 117, 119, 121, 137, 139, 141, 143, 145, 147, or 179.
  • the application provides the biepitopic tetravalent antibody, wherein the antibody backbone comprises Nimotuzumab, and wherein the scFv domain comprises a binding domain derived from the variable region of ABT-806 or humanized ABT-806. In one embodiment, the scFv domain comprises the variable regions of ABT-806. In one embodiment, the antibody heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.5, 15, 45, 47, 49, 51, 53, 55, or 85.
  • the antibody light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.7, 57, 59, 61, 63, 65, 67, 69, 71, or 73.
  • the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.205.
  • the antibody VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.207.
  • the scFv VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.209. In one embodiment, the scFv VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO.211. In one embodiment, the antibody VH domain comprises 3 complementary determining regions (CDRs) of SEQ ID NO.205. In one embodiment, the antibody VL domain comprises 3 CDRs of SEQ ID NO.207. In one embodiment, the scFv VH domain comprises 3 CDRs of SEQ ID NO.209.
  • CDRs complementary determining regions
  • the scFv VL domain comprises 3 CDRs of SEQ ID NO.211.
  • the biepitopic tetravalent antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO. 5, 7, 15, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 85.
  • the application provides a dematured Cetuximab monoclonal antibody, comprising a light chain having a light chain variable (VL) domain, and a heavy chain having a heavy chain variable (VH) domain, wherein the VL domain and the VH domain forms a Fab region, and wherein the monoclonal antibody comprises a Cetuximab dematuration mutation (sequential numbering) selected from VH-Y101, VH-Y102, VH-D103, VH-Y104, VL-N92, and a combination thereof.
  • VL light chain variable
  • VH heavy chain variable
  • the Cetuximab dematuration mutation comprises VH-Y101A, VH-Y101W, VH-Y102A, VH-D103F, VH-D103W, VH-D103Y, VH-Y104L, VL- N92F, VL-N92K, or a combination thereof.
  • the dematured Cetuximab monoclonal antibody is non-humanized.
  • the antibody VH domain comprises 3 complementary determining regions (CDRs) having an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of sequence identity to SEQ ID NO.247, 251, 253, 259, 261, 263, 265, or 267.
  • the antibody VL domain comprises 3 CDRs having an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of sequence identity to SEQ ID NO.243 or 269.
  • the heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of sequence identity to SEQ ID NO.1, 155, 157, 159, 161, 163, 165, 167, or 169.
  • the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of sequence identity to SEQ ID NO.247, 251, 253, 259, 261, 263, 265, or 267.
  • the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of sequence identity to SEQ ID NO. 3, 151, or 153.
  • the VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of sequence identity to SEQ ID NO.243 or 269.
  • the dematured Cetuximab monoclonal antibody is humanized.
  • the antibody VH domain comprises 3 complementary determining regions (CDRs) having an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of sequence identity to SEQ ID NO. 245, 249, 255, or 257.
  • the antibody VL domain comprises 3 complementary determining regions (CDRs) having an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of sequence identity to SEQ ID NO.241.
  • the heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of sequence identity to SEQ ID NO.123, 127, 129, 131, or 133.
  • the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of sequence identity to SEQ ID NO.245, 249, 255, or 257.
  • the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of sequence identity to SEQ ID NO. 99 or 109.
  • the antibody VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of sequence identity to SEQ ID NO. 241.
  • the application provides a multispecific antibody, comprising an antibody backbone, and at least one scFv domain linked to the antibody backbone, wherein the antibody backbone comprises the dematured Cetuximab monoclonal antibody as disclosed herein.
  • the application provides a multispecific antibody, comprising an antibody backbone, and at least one scFv domain linked to the antibody backbone, wherein the scFv domain comprise variable regions of dematured Cetuximab monoclonal antibody as disclosed herein.
  • Humanized anti-EGFRvIII monoclonal antibodies In one aspect, the application provides a humanized anti-EGFRvIII monoclonal antibody, comprising a light chain having a light chain variable (VL) domain, and a heavy chain having a heavy chain variable (VH) domain, wherein the VL domain and the VH domain forms a Fab region.
  • the monoclonal antibody is ABT0806 or humanized ABT-806.
  • the heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of the sequence identity to SEQ ID NO.171, 149, or 177.
  • the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of the sequence identity to SEQ ID NO.111, 173, or 175.
  • the VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of the sequence identity to SEQ ID NO. 225, 229, or 233.
  • the VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of the sequence identity to SEQ ID NO.227, 231, or 235.
  • the application provides a multispecific antibody, comprising an antibody backbone, and at least one scFv domain linked to the antibody backbone, wherein the antibody backbone comprises the humanized anti-EGFRvIII monoclonal antibody as disclosed herein.
  • the application provides a multispecific antibody, comprising an antibody backbone, and at least one scFv domain linked to the antibody backbone, wherein the scFv comprises variable regions of the humanized anti-EGFRvIII as disclosed herein.
  • the application provides a tetravalent antibody having a binding affinity to EGFR, comprising an antibody light chain having an antibody light chain variable (VL) domain, an antibody heavy chain having an antibody heavy chain variable (VH) domain, and a scFv domain having a scFv light chain variable (VL) domain and scFv heavy chain variable (VH) domain, wherein the antibody VL domain and the antibody VH domain forms a Fab region, wherein the scFv domain is linked to at least one end of the antibody light chain or the antibody heavy chain.
  • the antibody VH domain comprises an amino acid sequence having a SEQ ID NO. 201.
  • the antibody VL domain comprises an amino acid sequence having a SEQ ID NO.203. In one embodiment, the antibody heavy chain comprises an amino acid sequence having a SEQ ID NO. 89. In one embodiment, the antibody light chain comprises an amino acid sequence having a SEQ ID NO.3. In one embodiment, the scFv domain comprises a light chain variable (VL) domain having an amino acid sequence having a SEQ ID NO. 203. In one embodiment, the scFv domain comprises a heavy chain variable (VH) domain having an amino acid sequence having a SEQ ID NO.201. In one embodiment, the antibody heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% of the sequence identity to SEQ ID NO. 89.
  • the antibody light chain comprises SEQ ID NO.3.
  • the antibody VH comprises SEQ ID NO.201.
  • the antibody VL comprises SEQ ID NO.203.
  • the antibody scFv VH comprises SEQ ID NO.201.
  • the antibody scFv VL comprises SEQ ID NO.203.
  • the antibody VH comprises 3 complementary determining regions (CDRs) of SEQ ID NO.201.
  • the antibody VL comprises 3 CDRs of SEQ ID NO. 203.
  • the antibody scFv VH comprises 3 CDRs: SEQ ID NO. 201.
  • the antibody scFv VL 3 CDRs comprises SEQ ID NO.203.
  • the tetravalent antibody is represented by SI-95X42, as shown in the example below.
  • the application provides a tetravalent antibody having a binding affinity to EGFRvIII, comprising an antibody light chain having an antibody light chain variable (VL) domain, an antibody heavy chain having an antibody heavy chain variable (VH) domain, and a scFv domain having a scFv light chain variable (VL) domain and scFv heavy chain variable (VH) domain, wherein the antibody VL domain and the antibody VH domain forms a Fab region, wherein the scFv domain is linked to at least one end of the antibody light chain or the antibody heavy chain.
  • VL antibody light chain variable
  • VH antibody heavy chain variable
  • the antibody VH domain comprises an amino acid sequence having a SEQ ID NO. 209. In one embodiment, the antibody VL domain comprises an amino acid sequence having a SEQ ID NO.211. In one embodiment, the antibody heavy chain comprises an amino acid sequence having a SEQ ID NO.87. In one embodiment, the antibody light chain comprises an amino acid sequence having a SEQ ID NO. 11. In one embodiment, the scFv domain comprises a light chain variable (VL) domain having an amino acid sequence having a SEQ ID NO. 211. In one embodiment, the scFv domain comprises a heavy chain variable (VH) domain having an amino acid sequence having a SEQ ID NO.209. In one embodiment, the antibody heavy chain comprises SEQ ID NO. 87.
  • the antibody light chain comprises SEQ ID NO. 11.
  • the antibody VH domain comprises SEQ ID. NO.209.
  • the antibody VL domain comprises SEQ ID NO.211.
  • the antibody scFv VH domain comprises SEQ ID NO.209.
  • the antibody scFv VL domain comprises SEQ ID NO.211.
  • the antibody VH domain comprises 3 complementary determining regions (CDRs) of SEQ ID NO.209.
  • the antibody VL domain comprises 3 CDRs of SEQ ID NO.211.
  • the antibody scFv VH domain comprises 3 CDRs of SEQ ID NO.209.
  • the antibody scFv VL domain comprises 3 CDRs of SEQ ID NO. 211.
  • the intraspecific antibody is represented by SI-95X41, as shown in the example below.
  • the application provides isolated nucleic acid sequences encoding the biepitopic tetravalent antibody, monoclonal antibodies, tetraspecific antibodies, or multispecific antibodies as disclosed herein.
  • the application provides expression vectors comprising the nucleic acid sequences as disclosed herein, wherein the vector is expressible in a cell.
  • the application provides host cells comprising the nucleic acid as disclosed herein.
  • the host cell comprises the expression vector as disclosed herein.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the application provides methods of producing the antibodies as disclosed herein. In one embodiment, the method includes culturing the host cell disclosed herein so that the disclosed antibody is produced. In one embodiment, the antibody is a biepitopic tetravalent antibody. In one embodiment, the antibody is monoclonal antibodies. In one embodiment, the antibody is tetraspecific antibodies. In one embodiment, the antibody is multispecific antibodies. In a further aspect, the application provides immunoconjugates comprising the biepitopic tetravalent antibody as disclosed herein and a cytotoxic agent.
  • the cytotoxic agent comprises a chemotherapeutic agent, a growth inhibitory agent, a toxin, or a radioactive isotope.
  • the application provides a pharmaceutical composition, comprising the antibody as disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further includes radioisotope, radionuclide, a toxin, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.
  • the antibody is a biepitopic tetravalent antibody.
  • the antibody is monoclonal antibodies.
  • the antibody is tetraspecific antibodies.
  • the antibody is multispecific antibodies.
  • the application provides pharmaceutical composition, comprising the immunoconjugate as disclosed herein and a pharmaceutically acceptable carrier.
  • the application provides method of treating a subject with a cancer, comprising administering to the subject an effective amount of the biepitopic tetravalent antibody as disclosed herein.
  • the cancer comprises cells expressing EGFR, EGFRvIII or both.
  • the cancer comprises breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, non-small lung cell cancer, small cell lung cancer, glioma, esophageal cancer, nasopharyngeal cancer, kidney cancer, gastric cancer, liver cancer, bladder cancer, cervical cancer, brain cancer, lymphoma, leukemia, myeloma.
  • the method of treating further includes co-administering an effective amount of a therapeutic agent.
  • the therapeutic agent comprises an antibody, a chemotherapy agent, an enzyme, or a combination thereof.
  • the therapeutic agent comprises capecitabine, cisplatin, trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testolactone, vorozole, formestane, fadrozole, letrozole, erlotinib, lafatinib, dasatinib, gefitinib, imatinib, pazopinib, lapatinib, sunitinib, nilotinib, sorafenib, nab-palitaxel, a derivative or a combination thereof.
  • the subject is a human.
  • the application provides a solution comprising an effective concentration of the biepitopic tetravalent antibody as disclosed herein, wherein the solution is blood plasma in a subject.
  • Figure 1 depicts the configurations of biepitopic tetravalent antibodies capable of targeting two epitopes of EGFR comprising two anti-EGFR Fab domains and two anti-EGFRvIII scFv domains (A) or two anti-EGFRvIII Fab domains and two anti-EGFR scFv domains (B), wherein the scFv domain may be linked to N- or C-terminus of heavy or light chain of the antibody, and the binding domain may be derived from Cetuximab (which may be humanized and/or dematured) or Nimotuzumab in combination with ABT-806 (which may be humanized);
  • Figure 2 depicts a structural alignment of Cetuximab (PDB 1YY9), Panitumumab (PDB 5SX4)
  • scFv domains were appended to various termini of antibody heavy and light chains.
  • an EGFRvIII-targeting scFv was attached to the N- or C-terminus (via the heavy or light chain) of an antibody targeting EGFR ( Figure 1A, 1B, 1C, and 1D).
  • an EGFRwt-targeting scFv was attached to the N- or C-terminus (via the heavy or light chain) of an antibody targeting EGFRvIII.
  • the present application relates to methods of making and using bispecific tetravalent antibodies, specifically, biepitopic tetravalent antibodies.
  • epipe defines the region of an antigen that binds to an antibody.
  • antibody is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, as well as antibody fragments (e.g., Fab, F(ab′) 2 , and Fv), so long as they exhibit the desired biological activity.
  • the antibody may be monoclonal, polyclonal, chimeric, single chain, bispecific or bi-effective, human and humanized antibodies as well as active fragments thereof.
  • active fragments of molecules that bind to known antigens include Fab, F(ab′)2, scFv and Fv fragments, including the products of a Fab immunoglobulin expression library and epitope-binding fragments of any of the antibodies and fragments mentioned above.
  • antibody may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain a binding site that immunospecifically bind an antigen.
  • the immunoglobulin can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses of immunoglobulin molecule.
  • the antibody may be whole antibodies and any antigen-binding fragment derived from the whole antibodies.
  • a typical antibody refers to heterotetrameric protein comprising typically of two heavy (H) chains and two light (L) chains. Each heavy chain is comprised of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain is comprised of a light chain variable domain (abbreviated as VL) and a light chain constant domain.
  • VH and VL regions can be further subdivided into domains of hypervariable complementarity determining regions (CDR), and more conserved regions called framework regions (FR).
  • CDR hypervariable complementarity determining regions
  • FR framework regions
  • Each variable domain is typically composed of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino-terminus to carboxy-terminus.
  • FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino-terminus to carboxy-terminus.
  • binding regions that interacts with the antigen.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against one common antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • the term “monoclonal antibody” refers to the group of monoclonal monospecific antibodies during the early year of development.
  • the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler & Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567).
  • the modifier “monoclonal” of the term “monoclonal multi-specific antibody” is often understandably omitted.
  • the monoclonal antibodies may include “chimeric” antibodies (immunoglobulins) 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, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).
  • chimeric antibodies immunoglobulins 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
  • Monoclonal antibodies can be produced using various methods including mouse hybridoma or phage display (see Siegel. Transfus. Clin. Biol.9:15-22 (2002) for a review) or from molecular cloning of antibodies directly from primary B cells (see Tiller. New Biotechnol.28:453- 7 (2011)).
  • the antibody variable genes were isolated using recombinant DNA techniques and the resulting antibodies were expressed recombinantly and further screened for desired features such as ability to inhibit the binding of EGFR in its wild type or mutant forms. This general method of antibody discovery is similar to that described in Seeber et al. PLOS One. 9:e86184 (2014).
  • monoclonal antibody genes may be humanized, engineered, combined, and otherwise modified to generate various types of multispecific antibodies.
  • Cetuximab anti-EGFR
  • ABT-806 anti-EGFRvIII
  • the term “antigen- or epitope-binding portion or fragment” refers to fragments of an antibody that are capable of binding to an antigen such as EGFR in the present application. These fragments may be capable of the antigen-binding function and additional functions of the intact antibody.
  • binding fragments include but are not limited to a single-chain Fv fragment (scFv) consisting of the VL and VH domains of a single arm of an antibody connected in a single polypeptide chain by a synthetic linker or a Fab fragment which is a monovalent fragment consisting of the VL, constant light (CL), VH and constant heavy 1 (CH1) domain.
  • scFv single-chain Fv fragment
  • Fab fragment which is a monovalent fragment consisting of the VL, constant light (CL), VH and constant heavy 1 (CH1) domain.
  • Antibody fragments can be even smaller sub-fragments and can consist of domains as small as a single CDR domain, in particular the CDR3 regions from either the VL and/or VH domains (for example see Beiboer et al., J. Mol. Biol. 296:833-49 (2000)).
  • Antibody fragments are produced using conventional methods known to those skilled in the art.
  • the antibody fragments can be screened for utility using the same techniques employed with intact antibodies.
  • the “antigen- or epitope-binding fragments” can be derived from an antibody of the present disclosure by several art-known techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like.
  • an enzyme such as pepsin
  • Papain digestion of antibodies produces two identical antigen binding fragments, called “Fab” fragments, each with a single antigen binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily.
  • Pepsin treatment yields an F(ab′)2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • the Fab fragment may contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • “Fv” is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody.
  • variable domain or half of an Fv comprising only three CDRs specific for an antigen
  • ⁇ The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , delta, epsilon, ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • a “humanized antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s).
  • framework support residues may be altered to preserve binding affinity.
  • polypeptide “peptide”, and “protein”, as used herein, are interchangeable and are defined to mean a biomolecule composed of amino acids linked by a peptide bond.
  • the terms “a”, “an” and “the” as used herein are defined to mean “one or more” and include the plural unless the context is inappropriate.
  • isolated is meant a biological molecule free from at least some of the components with which it naturally occurs. "Isolated,” when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step.
  • an “isolated antibody,” refers to an antibody which is substantially free of other antibodies having different antigenic a binding specificity.
  • “Recombinant” means the antibodies are generated using recombinant nucleic acid techniques in exogeneous host cells.
  • the term “antigen” refers to an entity or fragment thereof which can induce an immune response in an organism, particularly an animal, more particularly a mammal including a human.
  • the term includes immunogens and regions thereof responsible for antigenicity or antigenic determinants.
  • immunogenic refers to substances which elicit or enhance the production of antibodies, T-cells or other reactive immune cells directed against an immunogenic agent and contribute to an immune response in humans or animals.
  • an immune response occurs when an individual produces sufficient antibodies, T-cells and other reactive immune cells against administered immunogenic compositions of the present disclosure to moderate or alleviate the disorder to be treated.
  • immunogenicity refers to an ability of therapeutics, such as a non-humanized antibody, to trigger an undesirable immune response against the therapeutics.
  • Specific binding or “specifically binds to” or is "specific for" a particular antigen or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity.
  • specific binding can be determined by competition with a control molecule that is similar to the target.
  • Specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KD for an antigen or epitope of at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, alternatively at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M, or greater, where KD refers to a dissociation rate of a particular antibody-antigen interaction.
  • an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000- , 10,000- or more times greater for a control molecule relative to the antigen or epitope.
  • specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction.
  • “Homology” between two sequences is determined by sequence identity.
  • sequence identity preferably relates to the percentage of the nucleotide residues of the shorter sequence which are identical with the nucleotide residues of the longer sequence. Sequence identity can be determined conventionally with the use of computer programs. The deviations appearing in the comparison between a given sequence and the above-described sequences of the disclosure may be caused for instance by addition, deletion, substitution, insertion, or recombination.
  • the term “dematuration” refers to a process by which a mature antibody (such as Cetuximab in this application) is engineered to have a mutation in its binding domain (such as a point mutation).
  • ADCC antibody-dependent cellular cytotoxicity
  • Fc receptor-bearing effector cells recognize and kill antibody-coated target cells that express tumor-associated or pathogen-derived antigens on their surface.
  • ADCC is widely used for characterizing and comparing therapeutic candidates of monoclonal antibodies.
  • Classical ADCC is mediated by natural killer (NK) cells.
  • NK natural killer
  • EXAMPLES Example 1 Engineering biepitopic tetravalent antibodies targeting EGFR Genes encoding antibody heavy and light chains (preceded by Kozak and secretory signal peptide) were cloned into pTT5 vector using standard molecular biology techniques. Antibodies and bispecific antibodies were expressed by transiently transfecting the expression plasmids for heavy and light chains in the ExpiCHO system (Thermo Fisher). Briefly, 10 ⁇ g of each expression plasmid was brought to 1ml with OptiPRO SFM medium. 1ml of OptiPRO SFM medium containing 80ul Expifectamine CHO reagent was added to the DNA and incubated at room temperature for 2.5 minutes.
  • the resulting mixture was then added to 25ml ExpiCHO cells at 6x10 6 cells/ml in a 125ml Erlenmeyer flask and incubated at 37°C, 5% CO 2 , 150rpm.
  • Cells were fed with 8.75ml ExpiCHO feed and 150 ⁇ l of CHO enhancer at 24 hours post-transfection and temperature shifted to 32°C, 5% CO 2 , 150rpm.
  • Cells were fed again at 48 hours post-transfection with 8.75ml ExpiCHO feed. Culture supernatant was harvested 9 days post-transfection, spun for 1 hour at 4500rpm to pellet the cells and then passed through a 0.2mm filter.
  • Expression titer was quantitated using biolayer interferometry on an Octet384 system with protein A sensors and a standard curve prepared with purified bispecific antibody protein. Proteins were purified from the harvested supernatant using a 5-ml MabSelect PrismA protein A column (GE Healthcare) equilibrated in phosphate-buffered saline on the AKTA Avant system. The supernatant was then passed over the column at a flow rate of 5 ml/min and washed with 25 ml PBS (125 mM sodium phosphate, 137 mM sodium chloride, pH 6.8). Protein of interest was then eluted by passing 15 ml of 50 mM sodium acetate, pH 3.5 over the column.
  • PBS 125 mM sodium phosphate, 137 mM sodium chloride, pH 6.8
  • the eluted protein was immediately neutralized by addition of 0.5ml 1M Tris-Cl, pH8.0.
  • proteins were analyzed by analytical SEC using an ACQUITY UPLC® Protein BEH SEC 200 ⁇ , 4.6mm x 150mm, 1.7 ⁇ m column controlled by a Waters Acquity UPLC H-Class. 10 ⁇ g of protein was injected into a mobile phase of PBS and flowed at 0.3 ml/min for 10 minutes.
  • Proteins were further purified by preparative SEC using a Superdex Increase 10/300 GL column in mobile phase of 25 mM sodium acetate, 125 mM NaCl, pH 5.5 on the AKTA Pure system, and ultimately buffer-exchanged into 25 mM sodium acetate, 125 mM NaCl, 10% sucrose, pH 5.5. Final samples contained >95% protein of interest as assessed by analytical SEC and were used for subsequent assays. Biolayer interferometry (Octet) binding assays (affinity) were performed on an Octet384 instrument to quantify binding kinetics of bispecific antibodies to EGFR and EGFRvIII.
  • Antibody was captured to anti-human Fc (AHC) sensor tips by loading for 180 seconds at 5 ⁇ g/ml. After a 60-second baseline step, a 180-second association phase with serial dilutions (0-100 nM; 1:2 dilution factor) of His-tagged EGFR or EGFRvIII (expressed and purified in-house) in assay buffer (PBS containing 0.1% BSA, 0.05% Tween20) was performed, followed by a 300-second dissociation phase in assay buffer. Regeneration was achieved using 10 mM glycine, pH 1.5. Binding curves were globally fit to a 1:1 model to extract the dissociation constants, K D , and kinetic association and dissociation rates.
  • AHC anti-human Fc
  • Biolayer interferometry (Octet) binding assays were performed on an Octet384 instrument to quantify bivalent binding kinetics of bispecific antibodies to immobilized EGFR and EGFRvIII.
  • Biotinylated EGFR or EGFRvIII was captured to streptavidin (SA) sensor tips by loading for 120 seconds at 1 ⁇ g/ml.
  • SA streptavidin
  • Binding curves were globally fit to a 1:1 model to extract the dissociation constants, KD, and kinetic association and dissociation rates.
  • Biolayer interferometry (Octet) was used for epitope binning studies.
  • Biotinylated EGFR- WT (Acro EGR-H82E3) or EGFRvIII (Acro EGR-H82E0) was immobilized at 1 ug/ml for 180 seconds onto streptavidin sensors in assay buffer (PBS containing 0.1% BSA, 0.05% Tween20). After a 30- second baseline, association with 100 nM of the first antibody was performed for 300 seconds. Finally, association with 100 nM of the second antibody was performed for 300 seconds.
  • Example 2 Expression titer Protein stability is a key parameter defined by the difference in free energy between the folded and unfolded states. For protein therapeutics, stability may impact immunogenicity, pharmacokinetics, and even efficacy. The reduction of aggregation can help to develop therapeutics that are easier to manufacture and safer for patients. In addition, expression efficiency and protein yield directly determine the cost of protein therapeutics. Manufacturing costs can be reduced significantly if proteins can be efficiently expressed to reach higher titers and increased yield of purified protein. After transient expression in ExpiCHO cells, titer was quantitated using biolayer interferometry (Table 2).
  • Monoclonal antibodies (EGFR or EGFRvIII) containing simple structures were produced with the highest efficiency, whereas bispecific (EGFR x EGFRvIII, SI-95X1-31, SI- 95X35-39) antibodies were produced with variable efficiency depending primarily on the composition of the binding domains.
  • Bispecific antibodies with nimotuzumab scFv (SI-95X4-16) were produced with significantly lower titer, indicative of the instability of the nimotuzumab VH and VL with this scFv format.
  • Example 3 Stability by analytical size-exclusion chromatography Immediately following first-step protein A purification, bispecific EGFR x EGFRvIII antibodies were evaluated for stability and aggregation by analytical size-exclusion chromatography on a Waters UPLC system (Table 2).
  • Antibodies containing nimotuzumab scFv generally showed low signal due to low expression and tended to have a high abundance of high molecular weight (HMW) or low molecular weight (LMW) contaminants in addition to the main peak corresponding to protein of interest (POI). This suggests that the nimotuzumab scFv is not stable when incorporated into the bispecific antibody.
  • the other bispecific antibodies showed on average more protein of interest, and less HMW and LMW species.
  • the proteins with C-terminal scFvs fused with the heavy chain (SI-95X1-3) showed the highest %POI, which is indicative of good stability.
  • Example 4 Octet binding to EGFR WT (EGFRwt) Biolayer interferometry was used to assess the affinity of bispecific antibodies for wild- type EGFR (Table 3). Cetuximab and nimotuzumab antibodies showed strong binding to wild-type EGFR, as demonstrated by response values of 0.75 and 0.32 nm respectively, whereas ABT-806 (an EGFRvIII-specific antibody) showed a much lower response of 0.07 nm. Most EGFR x EGFRvIII bispecific antibodies showed strong binding to wild-type EGFR, with the exception of those containing nimotuzumab scFv and ABT-806 Fab (SI-95X4-16) which showed low binding.
  • nimotuzumab has reduced function when present in scFv format.
  • nimotuzumab was in the Fab position (SI-95X2, SI-95X17-31), or when Cetuximab was in the Fab (SI-95X1, SI-95X37-39) or scFv (SI-95X3, SI-95X35-36) position, good binding to wild-type EGFR was observed.
  • Affinity data showed that antibodies with the Cetuximab domain had stronger affinity (lower KD) than those with the nimotuzumab domain.
  • Example 5 Octet binding to EGFRvIII Biolayer interferometry was used to assess the affinity of bispecific antibodies for EGFRvIII (Table 4). As expected, all monoclonal and bispecific antibodies targeting wild-type EGFR and/or EGFRvIII showed detectable binding to EGFRvIII, consistent with both binding epitopes being present in the EGFRvIII protein. Cetuximab had stronger binding response and affinity compared to nimotuzumab, whereas binding of ABT-806 was intermediate.
  • bispecific antibodies containing nimotuzumab scFv and ABT-806 Fab (SI-95X4-16) the observed binding was similar to that of the ABT-806 monoclonal antibody, indicating that nimotuzumab scFv is not contributing extra functionality.
  • bispecific antibodies containing nimotuzumab Fab and ABT-806 scFv (SI-95X2, SI-95X17-31) had lower KD values and slower dissociation rates, in some cases showing enhanced affinity compared to the component mAbs.
  • the same affinity enhancement was observed for bispecific antibodies containing Cetuximab and ABT-806 domains (SI-95X1, SI- 95X3, SI-95X35-39).
  • Example 6 Epitope binning (EGFRwt epitope) Biolayer interferometry was used for epitope binning of a panel of monoclonal antibodies with wild-type EGFR (Table 5). Antibodies included EGFR-specific Cetuximab (Cetu), Panitumumab (Pani), Nimotuzumab (Nimo), and Necitumumab (Neci), as well as EGFRvIII-specific ABT-806.
  • the four EGFR-targeting antibodies had detectable, but decreased binding if Nimotuzumab was used as the first antibody. This is likely because Nimotuzumab has slow association kinetics and was not able to completely saturate the EGFR in the first 300-second step.
  • the data confirm that ABT-806 does not bind significantly to wild-type EGFR and indicate that all four EGFR-targeting antibodies (Cetuximab, Panitumumab, Nimotuzumab, Necitumumab) share a conserved epitope. This latter conclusion is supported by other studies showing that panitumumab, Cetuximab, and Nimotuzumab have overlapping epitopes within EGFR domain III.
  • ABT-806 when ABT-806 was used as the second antibody, a large additional response was observed.
  • the lack of competitive binding between ABT-806 and the other antibodies is an indication that ABT-806 targets a unique epitope on EGFRvIII, and that ABT-806 and other EGFR- binding antibodies may simultaneously interact with different epitopes on the EGFRvIII protein. That ABT-806 binds a distinct epitope of EGFRvIII from EGFR-targeting antibodies is consistent with ABT-806 binding a conformation of the EGFR protein that is restricted to mutant EGFRvIII and not present on wild-type EGFR.
  • Example 8 Generation and characterization of humanized ABT-806 To increase the humanness of the ABT-806 variable regions and decrease the potential for immunogenicity, the mouse VH and VK domains were converted to a more human framework. All versions used scFv models generated from the antibody modeling feature of Discovery Studio based on the sequence of ABT-806 variable domains. All humanized versions were designed using the Discovery Studio 2022 suite. VH1, VH2, VK1, and VK2 were designed using the “Predict Humanizing Mutations protocol” with the identity threshold set to 50, the frequent residue substitution tolerance set to 20, the germline substitution tolerance set to 0, and the exclusion of substitutions of Kabat CDR residues, IMGT CDR residues, Vernier zone residues, and human germline residues.
  • This protocol is based exclusively on the amino acid sequence of ABT-806 as the query sequence.
  • VH1 and VK1 were generated based on germline substitutions, whereas versions VH2 and VK2 used frequent residue substitutions.
  • VH3 and VK3 were designed using an input Fv model for ABT806, with “calculate mutation energy” set to true (CHARMm forcefield) in order to generate the “best single mutations” sequences.
  • the query structure was a model for ABT-806 generated by Discovery Studio’s Antibody Modeling Cascade.
  • the input sequences were ABT-806 VH and VL.
  • the CDR loop definition was set to Honegger and the maximum templates per loop was set to 3, with the optimization level set to high.
  • V1 VH1, VK1
  • V2 VH1, VK2
  • V3 VH1, VK3
  • V4 VH2, VK1
  • V5 VH2, VK2
  • V6 VH2, VK3
  • V7 VH3, VK1
  • V8 VH3, VK2
  • V9 VH3, VK3
  • Heavy chain and light chain genes were cloned and expressed as in Example 1, and then antibodies were evaluated for stability and functional properties. After transient expression in ExpiCHO cells, titer was quantitated using biolayer interferometry (Table 7). The optimal humanized version was selected (partially) based on overall expression.
  • Antibodies were purified by protein A and SEC chromatography as described in Example 1. Following protein A purification, many antibodies tended to have a high abundance of high molecular weight (HMW) or low molecular weight (LMW) contaminants in addition to the main peak corresponding to protein of interest (POI). This suggests that these antibodies are less stable, prefer to form other species (HC Dimers), and/or are more prone to aggregation. V3 and V9 seem to be the most stable humanizations based on the above metrics (Table 7). Samples used for subsequent studies were purified to >95% purity by preparative SEC.
  • HMW high molecular weight
  • LMW low molecular weight
  • Biolayer interferometry (Octet) binding assays were performed on an Octet384 instrument to quantify binding kinetics of humanized antibodies to EGFR and EGFRvIII, as described in Example 1.
  • Table 8 shows that all monoclonal antibodies targeting EGFRvIII displayed detectable binding to EGFRvIII. Although similar binding kinetics were shown across all antibodies, the humanized V3 and V9 antibodies appeared to have higher responses rivaling that of ABT-806 and outperforming competitor Depatuxizumab, AbbVie’s humanized ABT-806 (purified in house).
  • purified primary antibodies were added to EGFR- or EGFRvIII- or uPAR-transfected CHO cells.
  • residues of interest are as follows (sequential numbering): from the light chain W94, N91, and N92; from the heavy chain W52, N56, Y101, Y102, D103, Y104. Each residue of interest was individually mutated to one of the 20 naturally occurring amino acids. In total 180 (9 residues * 20 amino acids) independent mutations were generated in silico.
  • the effect of a mutation on Cetuximab stability was calculated using the Discovery Studio Calculate Mutation Energy (Stability) function while the effect of the mutation on EGFR binding was calculated using the Calculate Mutation Energy (Binding) function with EGFR set as the ligand molecule.
  • the stability function calculates the difference in energy between the WT Cetuximab and the mutant Cetuximab ( ⁇ G Stability)
  • Codon-optimized coding regions preceded by Kozak and secretory signal peptide sequences were cloned into the pTT5 vector using standard molecular biology techniques. Antibodies were expressed by transiently transfecting the light chains and heavy chains in the ExpiCHO system (ThermoFischer Scientific) as described in Example 1 but scaling down to a culture volume of 2 ml in 6-well plates. Proteins were purified from the harvested supernatant using a Captureem Protein A 24-Well Plate (TaKaRa). The supernatant was loaded onto the phosphate-buffered saline (PBS, 125 mM sodium phosphate, 137 mM sodium chloride, pH 6.8) equilibrated plate.
  • PBS phosphate-buffered saline
  • HMW molecular weight
  • POI protein of interest
  • Table 13 shows that most dematured Cetuximab variants have decreased binding affinity and/or decreased binding response to EGFR as compared to that of Cetuximab wild type, indicating that the selected mutations alter the ability of Cetuximab to bind EGFR.
  • the antibodies retain good avidity for immobilized EGFR.
  • T able 14 shows that most dematured Cetuximab variants also have decreased binding affinity and/or decreased binding response to EGFRvIII.
  • all selected dematured Cetuximab variants retained their ability to bind to EGFRvIII with similar binding kinetics. This data reinstates that Cetuximab binds to the same epitope on EGFRvIII and EGFR.
  • the variants had weaker affinity to EGFR and EGFRvIII, they retained good avidity for EGFR and EGFRvIII due to bivalent binding. Since avidity depends on the level of antigen on the surface, this result suggests that the dematured versions of Cetuximab may be more selective for tumors bearing a high amount of EGFR and/or EGFRvIII.
  • purified primary antibodies were added to EGFR-, EGFRvIII-, or uPAR-transfected CHO cells. A fluorescent secondary antibody against the primary antibody was used to measure antigen binding at the cell surface.
  • Table 15 shows that all tested antibodies (except HC: D103W) retained their binding properties to cells mimicking a cancer surface. The result indicates that these antibodies retain the binding behavior desirable for making antibody therapeutics.
  • Example 10 Generation and characterization of biepitopic tetravalent antibodies targeting EGFR Nimotuzumab has been approved for treatment of solid tumors in several countries after the approval of Cetuximab. At least one of its improvements is reduced skin toxicities, which seems to be correlated with its lower affinity for EGFR, i.e., a KD value of 9.1 nM versus 3.77 nM (Table 3).
  • Cetuximab variant kinetically is Y104L (KD 9.2 nM), whereas all other variants displayed even weaker EGFR affinity than Nimotuzumab, including Y101A (KD 67 nM), Y102A (97 nM), and N92F (34 nM).
  • Y101A KD 67 nM
  • Y102A 97 nM
  • N92F 34 nM
  • Biepitopic tetravalent antibodies targeting EGFR and EGFRvIII also known as anti-EGFR X EGFRvIII antibodies, were constructed by fusing scFvs onto the N- or C-terminus of antibody heavy or light chains.
  • Figure 1 depicts four exemplary structures, wherein the EGFR binding domain may be derived from humanized and/or dematured Cetuximab variants (hDC-EGFR) and formatted as either Fab or scFv while ABT-806 or humanized ABT-806 may be reciprocally formatted as either scFv or Fab.
  • hDC-EGFR dematured Cetuximab variants
  • ABT-806 or humanized ABT-806 may be reciprocally formatted as either scFv or Fab.
  • Table 16 lists four groups of configuration formats of anti-hDC-EGFR x hABT-806 antibodies, namely, SI-95X45-49, SI-95X52-56, SI-95X58-62, and SI-95X64-68.
  • Their EGFR binding domain was derived from humanized dematured Cetuximab variants (hDC-EGFR), namely, SI- 95m4-10.
  • SI-95X45-49 have a C-terminal fusion of an anti-ABT-806 scFv on the antibody heavy chain with an anti-hDC-EGFR in the Fab position
  • SI-95X52-56 contain an N-terminal fusion of an anti-hDC-EGFR scFv on the antibody heavy chain with an ABT-806 V9 in the Fab position
  • SI- 95X58-62 contain a C-terminal fusion of ABT-806 V9 scFv on the antibody light chain with an anti- hDC-EGFR in the Fab position
  • SI-95X64-68 contain a C-terminal fusion of an anti-hDC-EGFR scFv on the antibody heavy chain with an ABT-806 in the Fab position.
  • anti-EGFR x EGFRvIII antibodies were the same as or similar to those described in Example 1. After transient expression in ExpiCHO cells, titer was quantitated using biolayer interferometry (Table 17). As expected, these bispecific antibodies (SI-95X40-68) were produced with variable efficiency depending primarily on the composition and configurations of each binding domain. For example, molecules with an anti-hDC-EGFR scFv fused to the C-terminus of the antibody heavy chain (SI-95X63-68) seemed to express less than those with the with an anti- hDC-EGFR scFv fused to the N-terminus of the heavy chain (SI-95X51-56).
  • molecules with the anti-hDC-EGFR Y101A domain (SI-95X47, 53, 58, 65) tended to have high titers whereas those with an anti-hDC-EGFR Y104L domain (SI-95X46, 56, 61, 68) tended to have low titers.
  • anti-EGFR x EGFRvIII bispecific antibodies were evaluated for stability and aggregation by analytical size-exclusion chromatography on a Waters UPLC system (Table 17). The configuration of the molecule seemed to play the largest role in % protein of interest after first step purification. Large amount of high molecular weight aggregate demonstrates instability in the SI-95X57-62 molecules.
  • Example 11 Biepitopic tetravalent antibodies exert ADCC activity toward EGFRvIII-expressing cells
  • the ADCC assay was performed by using NK cells (1 donor) to co-culture with U87-MG-GFP cells expressing EGFRwt, U87-MG-EGFRvIII-NR (i.e., mKATE2+) cells expressing both EGFRwt and EGFRvIII, or the mixed culture of the two. Mixed cell cultures were seeded and started with 50:50 green/red signals.
  • SI-95m4 and SI-95m5 antibodies are monoepitopic bivalent antibodies targeting EGFRvIII and EGFRwt, respectively.
  • SI-95m4 failed to kill U87-MG-EGFRwt- GFP cells in the ADCC assay, confirming that this humanized anti-EGFRvIII antibody retained the exclusive binding specificity of the ABT-806 antibody.
  • SI-95m4 exerted ADCC activity to U87-MG-EGFRwt-GFP cells (i.e., the EGFRwt target cells, Figure 4A). This surprising result may be interpreted as a bystander effect of activated NK cells toward EGFRwt-expressing cells.
  • the panel of monoepitopic bivalent antibodies consisting of SI-95m6, SI-95m7, SI-95m8, SI-95m9, and SI-95m10 (Table 16), displayed a range of reduced ADCC activity toward EGFRwt-expressing cells when compared to the parental anti-hDC-EGFR antibody, SI-95m5 ( Figure 5A).
  • the EC50 value for the variants was increased up to 12-fold (for SI-95m6) compared to that of the parental mAb SI-95m5, indicating weaker potency.
  • SI-95m4 an anti-EGFRvIII control antibody for the assay, showed no activity.
  • SI-95m6 When the EGFRwt/EGFRvIII-expressing cells were used, SI-95m6, SI-95m9, and SI-95m10 exerted their ADCC activity at a level similar to the positive control antibody, SI-95m4 ( Figure 5B). Of this panel of antibodies, SI-95m7 displayed a right-shifted curve with 3-fold increased EC50, indicative of a characteristic reduction in ADCC activity. In the meantime, SI-95m8 no longer showed any activity toward EGFR ( Figure 5A and 5B), which is likely due to the specific D103Y mutation in its EGFR binding domain.
  • the panel of biepitopic tetravalent antibodies included SI-95X52, SI-95X53, SI-95X54, SI- 95X55, and SI-95X56, each of which contains an anti-EGFRvIII Fab binding domain, i.e., a hABT- 806 (V9) antibody backbone, and an anti-EGFRwt scFv binding domain.
  • the panel shared a common antibody backbone derived from their parental mAb, SI-94m4.
  • SI-95m10 corresponding to SI-95X52
  • SI-95m6 SI-95X53
  • SI-95m7 SI-95X54
  • SI-95m8 SI-95X55
  • SI-95m9 SI-95X56
  • the different configurations of biepitopic antibodies with regard to the geometry and distance of the anti-EGFR and anti-EGFRvIII binding domains may allow for varying capacities for this simultaneous binding mechanism on cells, which could impact the extent of ADCC against the EGFRwt-expressing tumor cells as well as EGFRwt/EGFRvIII-expressing tumor cells.
  • the synergetic effects of biepitopic tetravalent antibodies are likely influenced by the structural change as a result of humanization, dematuration, and/or configuration.
  • SI-95X55 exhibited improved potency to EGFRwt/EGFRvIII-expressing cells, as indicated by its EC50 (12.37 nM) as compared to that of either SI-95m4 (19.39 nM) or SI- 95X44 (14.72 nM) as listed in Figure 6B.
  • This observation indicates that even residual affinity/avidity to EGFRwt (Table 15) helped improve its potency of ADCC. It also demonstrates improvement of therapeutic index by decreasing potency against EGFRwt-bearing cells while increasing potency against EGFRwt/EGFRvIII-bearing cells.
  • Example 12 Biepitopic tetravalent antibodies targeting both EGFRwt and EGFRvIII expressing tumors.
  • Example 11 The results from Example 11 indicated that the ADCC activity of a dematured monoepitopic antibody may not predict the same for the biepitopic antibody harboring the same dematuration mutation.
  • SI-95m8, SI-95X55, and SI-95X67 harbor the same D103Y mutation.
  • the loss of ADCC potency against EGFRwt-expressing cells made SI-95X55 a candidate only for targeting EGFRvIII tumors.
  • the screen of biepitopic tetravalent antibodies that can efficiently target both EGFRwt and EGFRvIII expressing tumors was focused on those groups of antibodies harboring dematuration mutations, Y101A and Y102A.
  • the Y101A mutation is the common feature of dematured antibodies: SI-95m6 and SI- 95X53/SI-95X65, of which the antibody backbone of SI-95X65 was not humanized (Table 16).
  • the Y102A mutation is the common feature of dematured antibodies: SI-95m7, SI-95X54, and SI-95X66, of which the antibody backbone of SI-95X66 was not humanized and contains the anti-EGFR scFv domain on the opposite end of the heavy chain when compared to SI-95X54 (Table 16).
  • SI-95m7, SI-95X54, and SI-95X66 displayed right-shifted curves with incomplete killing
  • SI-95X54 responded with a complete killing curve of EGFRwt-expressing cells in a range between from 10 nM to 100nM (see table in Figure 7B).
  • SI-95X54 The right shift by SI-95X54 was measured at approximately 5- fold higher than the EC50 values of both SI-95m5 and SI-95X44.
  • SI-95X54 is a biepitopic tetravalent antibody having its scFv domain derived from the anti-hDC-EFGR variant with Y102A.
  • SI-95X54 unveils its EGFRvIII-biased cell killing while also killing EGFRwt tumors at least 5- fold higher EC50 value.
  • the panel of separate and mixed EGFRwt and EGFRvIII cells was used to evaluate bispecific candidates.
  • SI-95X44 displayed overlapping curves and similar ED50 values as expected ( Figure 8A).
  • a bispecific candidate such as SI-95X54 displayed a characteristic right-shift curve with a complete killing of mixed cells (Figure 8B).
  • SI- 95X54 is capable of efficiently killing a mixed EGFRwt and EGFRvIII cell population, which may mimic the heterogeneity of glioblastoma in a human patient, and that a reduction in its sensitivity to kill EGFRwt cells may imply a reduced toxicity to heathy tissues of the patient.
  • EGFRwt and EGFRvIII play complex and interactive roles in the genesis of brain tumors.
  • EGFR overexpression is widespread among the tumor cells in most cases of glioblastomas, whereas EGFRvIII typically shows patchy tumor positivity of only a minority of the tumor cells, when pan-EGFR antibodies are used for staining.
  • Such tumor heterogeneity remains as an unmet challenge for developing effective and meaningful treatment for brain tumors.
  • a therapeutic antibody simultaneously targeting both EGFRwt and EGFRvIII cells with EGFRvIII- biased binding may be a desirable solution.
  • this application provided the proof of concept of a therapeutic strategy by which biepitopic tetravalent antibodies have been generated to effectively eradicate mixed tumor cell populations expressing EGFRwt and/or EGFRvIII with lower toxicity to normal tissues.
  • Table 5 EGFRwt epitope binning of EGFRwt- and EGFRvIII-targeting antibodies. Table 6. EGFRvIII epitope binning of EGFRwt- and EGFRvIII-targeting antibodies Table 7. Characterization of humanized ABT-806 variants, such as the expression titer and purity as assessed by using analytical size-exclusion chromatography after protein A purification. Table 8. The binding affinity of humanized ABT-806 antibodies to EGFRvIII.
  • Table 17 Characterization of anti-EGFR x EGFRvIII bispecific antibodies comprising humanized dematured EGFR binding domain and humanized EGFRvIII binding domain, such as the expression titer and purity as assessed by using analytical size-exclusion chromatography after protein A purification.
  • Table 18 Binding kinetics of anti-EGFR x EGFRvIII biepitopic tetravalent antibodies comprising humanized dematured EGFR binding domain and humanized EGFRvIII binding domain to EGFR- WT Table 19. Binding kinetics of anti-EGFR x EGFRvIII bispecific antibodies comprising humanized dematured EGFR binding domain and humanized EGFRvIII binding domain to EGFRvIII Reference 1.

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Abstract

L'invention concerne un anticorps tétravalent biépitopique ayant une affinité de liaison à au moins deux épitopes EGFR. L'anticorps comprend un squelette d'anticorps et un domaine scFv lié au squelette d'anticorps. Les deux épitopes EGFR peuvent comprendre un épitope de type sauvage EGFR (EGFRwt) et un épitope EGFRvIII. L'anticorps présente une affinité de liaison plus forte avec l'épitope l'EGFRvIII qu'avec l'épitope EGFRwt.
PCT/US2023/073194 2022-08-31 2023-08-30 Anticorps tétravalent biépitopique ciblant l'egfr WO2024050439A2 (fr)

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