WO2022104697A1 - Modified egfr antibody with reduced affinity - Google Patents

Modified egfr antibody with reduced affinity Download PDF

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Publication number
WO2022104697A1
WO2022104697A1 PCT/CN2020/130417 CN2020130417W WO2022104697A1 WO 2022104697 A1 WO2022104697 A1 WO 2022104697A1 CN 2020130417 W CN2020130417 W CN 2020130417W WO 2022104697 A1 WO2022104697 A1 WO 2022104697A1
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Prior art keywords
seq
antibody
variable domain
amino sequence
light chain
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PCT/CN2020/130417
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French (fr)
Inventor
Bing Xia
Yuhong Zhou
Ziping Wei
Lixia CAO
Fangdun JIANG
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Bliss Biopharmaceutical (Hangzhou) Co., Ltd.
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Application filed by Bliss Biopharmaceutical (Hangzhou) Co., Ltd. filed Critical Bliss Biopharmaceutical (Hangzhou) Co., Ltd.
Priority to PCT/CN2020/130417 priority Critical patent/WO2022104697A1/en
Priority to AU2021381419A priority patent/AU2021381419A1/en
Priority to CN202180070808.5A priority patent/CN116419929B/en
Priority to US18/253,681 priority patent/US20240009318A1/en
Priority to EP21894025.2A priority patent/EP4247853A1/en
Priority to JP2023530648A priority patent/JP2023549933A/en
Priority to PCT/CN2021/131780 priority patent/WO2022105878A1/en
Priority to CA3199562A priority patent/CA3199562A1/en
Publication of WO2022104697A1 publication Critical patent/WO2022104697A1/en

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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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—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

  • Antibodies are key immune molecules acting against foreign pathogens.
  • mAb monoclonal antibody
  • the development of monoclonal antibody (mAb) technology resulted in widespread use of monoclonal antibodies in research, diagnosis and treatment of diseases.
  • the therapeutic use of first-generation mAb achieved success in the treatment of a variety of diseases, including cancer, autoimmune, and infectious diseases.
  • diseases, such as solid tumors have been shown to be quite resistant to antibody-based therapies.
  • ADCs Antibody Drug Conjugates
  • ADCs are mAbs chemically linked to active drugs, and therefore, have both the specific targeting of mAbs and the cancer-killing ability of cytotoxic drugs.
  • the ability to select specific mAbs-drug combination and advances in the linking the mAbs and drugs provides new possibilities to target cancers while minimizing exposure of healthy tissue.
  • ADCs have been approved by the FDA, including: ado-trastuzumab emtansine (Kadcyla TM ) , brentuximab vedotin (Adcetris TM ) , inotuzumab ozogamicin (Besponsa TM ) , gemtuzumab ozogamicin (Mylotarg TM ) , polatuzumab vedotin-piiq (Polivy TM ) , Enfortumab vedotin (Padcev TM ) , and Trastuzumab deruxtecan (Enhertu TM ) . All of them involve conjugation of a cytotoxic drug with a bivalent mAb. In addition to the seven ADC drugs that have been approved for marketing, a large number of ADCs are currently under clinical development.
  • the epidermal growth factor receptor (EGFR, also known as HER1 or c-erbB-1) is a 170 kDa transmembrane glycoprotein and a member of the tyrosine kinase family of cell surface receptors.
  • EGFR is abnormally activated in many epithelial tumors, including those in non-small cell lung cancer, breast cancer, colorectal cancer, head and neck cancers, and prostate cancer.
  • Abnormal activation of EGFR can arise from overexpression of the receptor, gene amplification, activating mutations, overexpression of receptor ligands, and/or loss of regulators of EGFR activity.
  • Interruption of EGFR signaling can inhibit the growth of EGFR-expressing tumors and improve the patient's condition.
  • Anti ⁇ EGFR antibodies exert antitumor effects by binding the receptor at the cell surface to interfere with ligand binding, which leads to the inhibition of its downstream signaling pathway.
  • Panitumumab is a clinically proven antibody that targeted EGFR potently and specifically.
  • clinical response to Panitumumab is often accompanied by significant toxicities due to normal tissue expression.
  • an isolated antibody which comprises (a1) a heavy chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 18, respectively, and (b1) a light chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23, respectively.
  • Another isolated antibody comprises: (b1) a heavy chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, and (b2) a light chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 23, respectively.
  • an isolated antibody which comprises: (a) a heavy chain having a variable domain comprising the amino sequence of SEQ ID NO: 1, and a light chain having a variable domain comprising the amino sequence of SEQ ID NO: 4; or (b) a heavy chain having a variable domain comprising the amino sequence of SEQ ID NO: 5, and a light chain having a variable domain comprising the amino sequence of SEQ ID NO: 6.
  • an isolated antibody which comprises: (a) a heavy chain comprising the amino sequence of SEQ ID NO: 7, and a light chain comprising the amino sequence of SEQ ID NO: 10; or (b) a heavy chain comprising the amino sequence of SEQ ID NO: 13, and a light chain comprising the amino sequence of SEQ ID NO: 10; or (c) a heavy chain comprising the amino sequence of SEQ ID NO: 11, and a light chain comprising the amino sequence of SEQ ID NO: 12.
  • the antibody of the present application can be monoclonal antibody.
  • the antibody of the present disclosure can be deemed as the antibody Panitumumab, which is the first fully human monoclonal antibody directed against the EGFR, being mutated at one amino acid on its heavy chain and one amino acid on its light chain.
  • the heavy chain mutation can be T103A while the light chain mutation is Y32A, or the heavy chain mutation can be T59A while the light chain mutation is D50A.
  • nucleic acid molecule encoding the antibody described herein an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector are also provided.
  • the present disclosure provides an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof that comprises the antibody described herein conjugated to a cytotoxic drug molecule by a chemical linker.
  • the cytotoxic drug can be selected from the group consisting of monomethyl auristatin E (MMAE) , monomethyl auristatin F (MMAF) , auristatin E, auristatin F, maytansine DM1 and DM4, maytansinol, sandramycin, pyrrolobenzodiazepine dimer, anthracyclines, calicheamicin, dolastatin 10, duocarmycin, doxorubicin, thailanstatin A, uncialamycin, amanitins, ricin, diphtheria toxin, eribulin, 131 I, interleukins, tumor necrosis factors, chemokines, and nanoparticles.
  • MMAE monomethyl auristatin E
  • the chemical linker linking the antibody portion and the cytotoxic drug can be cleavable or non-cleavable.
  • the linker comprises a PEGn spacer where n is between 1 and 20 (i.e., having 1 to 20 repeat units (CH 2 CH 2 O) ) .
  • the chemical linker further comprises a linker segment connected to the PEGn spacer.
  • the chemical linker comprises a linker segment but does not comprise a PEGn spacer.
  • the linker segment can be selected from the group consisting of 6-maleimidocaproyl (MC) , maleimidopropionyl (MP) , valine-citrulline (val-cit) , alanine-phenylalanine (ala-phe) , p-aminobenzyloxycarbonyl (PAB) , 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB) , Val-Cit-PABC, Phe-Lys (Fmoc) -PAB, Aloc-D-Ala-Phe-Lys (Aloc) -PAB-PNP, Boc-Phe- (Alloc) Lys-PAB-PNP, and perfluorophenyl 3- (pyridine-2-yldisulfanyl) propanoate, or combinations thereof.
  • MC 6-maleimidocaproyl
  • MP maleimidopro
  • Figure 1 shows schematic diagrams of interactions between paratope surfaces of EGFR protein and CDRs of panitumumab.
  • A Panitumumab light chain CDR interactions with the final ⁇ -strand of EGFR domain III. EGFR on top interactions with L1, L2 and L3 CDRs of the panitumumab on the bottom.
  • B Panitumumab heavy chain CDR interactions with the ⁇ -sheet surface of EGFR domain III that binds EGFR.
  • EGFR is shown on top with H1, H2, and H3 CDR regions, . Amino acids subject to mutation are circled.
  • Figure 2 shows characterization of antibodies according to some embodiments of the present invention.
  • Figure 3 shows binding Curves of certain antibodies according to embodiments of the present invention, among other antibodies, to EGFR.
  • FIG. 4 shows HIC profiles of two ADCs made of certain antibodies according to embodiments of the present invention. HIC profiles of two ADCs made of two different mutant antibodies.
  • Figure 5 shows cytotoxicity curves of certain ADCs according to embodiments of the present invention and comparative ADCs to EGFR-expressing cancer cells: (a) A431 cell, (b) NUGC3 cell.
  • the present disclosure provides antibodies based on modification of the amino acid sequences of panitumumab. Without losing the binding specificity to EGFR, the modified (or mutant) antibody has reduced affinity to the antigen, and has reduced toxicities on normal tissues. ADCs based on these antibodies are also provided. According to some embodiments, compared with the ADC made from the parent wild-type panitumumab and a same drug, ADCs made of the mutant antibodies retain comparable potency in terms of killing EGFR high-expressing tumor cells, while having significantly reduced potencies to EGFR low-expressing cells. Therefore, treatment with an ADC made of such mutant antibody can have a better therapeutic window with less on-target-off-tumor toxicities to EGFR low-expressing normal skin tissues that was associated with Panitumumab treatment.
  • monoclonal antibody refers to a preparation of antibody molecules of single molecular composition.
  • CDRs complementarity-determining regions
  • These systems and/or definitions have been developed and refined over a number of years and include Kabat, Chothia, IMGT, AbM, and Contact.
  • the Kabat definition is based on sequence variability and is commonly used.
  • the Chothia definition is based on the location of the structural loop regions.
  • the IMGT system is based on sequence variability and location within the structure of the variable domain.
  • the AbM definition is a compromise between Kabat and Chothia.
  • the Contact definition is based on analyses of the available antibody crystal structures.
  • An Exemplary system is a combination of Kabat and Chothia.
  • an isolated antibody which comprises (a1) a heaving chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 18, respectively, and (b1) a light chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23, respectively.
  • Another isolated antibody comprises: (b1) a heaving chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, and (b2) a light chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 23, respectively.
  • an isolated antibody which comprises: (a) a heavy chain having a variable domain comprising the amino sequence of SEQ ID NO: 1, and a light chain having a variable domain comprising the amino sequence of SEQ ID NO: 4; or (b) a heavy chain having a variable domain comprising the amino sequence of SEQ ID NO: 5, and a light chain having a variable domain comprising the amino sequence of SEQ ID NO: 6.
  • an isolated antibody which comprises: (a) a heavy chain comprising the amino sequence of SEQ ID NO: 7, and a light chain comprising the amino sequence of SEQ ID NO: 10; or (b) a heavy chain comprising the amino sequence of SEQ ID NO: 13, and a light chain comprising the amino sequence of SEQ ID NO: 10; or (c) a heavy chain comprising the amino sequence of SEQ ID NO: 11, and a light chain comprising the amino sequence of SEQ ID NO: 12.
  • DNA encoding an amino acid sequence variant of a starting polypeptide can prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the polypeptide. Variants of recombinant antibodies may be constructed also by restriction fragment manipulation or by overlap extension PCR with synthetic oligonucleotides. Mutagenic primers encode the cysteine codon replacement (s) . Standard mutagenesis techniques can be employed to generate DNA encoding such mutant engineered antibodies.
  • the present disclosure provides a nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any of the antibody described herein.
  • a host cell e.g., a CHO cell, a human embryonic kidney cell, a lymphocytic cell, or microorganisms, such as E. coli, and fungi, such as yeast
  • DNA encoding partial or full-length antibody of the present disclosure can be obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operatively linked to transcriptional and translational regulatory sequences.
  • operatively linked is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody genes. Such regulatory sequences are described, e.g., in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) ) .
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) , Simian Virus 40 (SV40) , adenovirus, e.g., the adenovirus major late promoter (AdMLP) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • nonviral regulatory sequences can be used, such as the ubiquitin promoter or ⁇ -globin promoter.
  • regulatory elements composed of sequences from different sources, such as the SR ⁇ promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8: 466-472) .
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody encoding DNA can be inserted into the expression vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody encoding DNA can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody encoding DNA.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein) .
  • the present disclosure provides an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof that comprises the antibody described herein conjugated to a cytotoxic drug molecule by a chemical linker.
  • the cytotoxic drug can be selected from the group consisting of monomethyl auristatin E (MMAE) , monomethyl auristatin F (MMAF) , auristatin E, auristatin F, maytansine DM1 and DM4, maytansinol, sandramycin, pyrrolobenzodiazepine dimer, anthracyclines, calicheamicin, dolastatin 10, duocarmycin, doxorubicin, thailanstatin A, uncialamycin, amanitins, ricin, diphtheria toxin, eribulin, 131 I, interleukins, tumor necrosis factors, chemokines, and nanoparticles.
  • MMAE monomethyl auristatin E
  • the chemical linker linking the antibody portion and the cytotoxic drug can be cleavable or non-cleavable.
  • the linker comprises a PEGn spacer where n is between 1 and 20 (i.e., having 1 to 20 repeat units (CH 2 CH 2 O) ) .
  • the chemical linker further comprises a linker segment connected to the PEGn spacer.
  • the chemical linker comprises a linker segment but does not comprise a PEGn spacer.
  • the linker segment can be selected from the group consisting of 6-maleimidocaproyl (MC) , maleimidopropionyl (MP) , valine-citrulline (val-cit) , alanine-phenylalanine (ala-phe) , p-aminobenzyloxycarbonyl (PAB) , 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB) , Val-Cit-PABC, Phe-Lys (Fmoc) -PAB, Aloc-D-Ala-Phe-Lys (Aloc) -PAB-PNP, Boc-Phe- (Alloc) Lys-PAB-PNP, and perfluorophenyl 3- (pyridine-2-yldisulfanyl) propanoate, or combinations thereof.
  • MC 6-maleimidocaproyl
  • MP maleimidopro
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more antibodies, ADCs or the pharmaceutically acceptable salts thereof, of the present invention, together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes pharmaceutically acceptable carriers, excipients or stabilizers. These include but are not limited solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and the like that are physiologically compatible.
  • composition may comprise one or more additional pharmaceutically active ingredients, such as another antibody, a drug, e.g., a cytotoxic or anti-tumor agent.
  • additional pharmaceutically active ingredients such as another antibody, a drug, e.g., a cytotoxic or anti-tumor agent.
  • the pharmaceutical compositions of the invention also can be administered in a combination therapy with, for example, another anti-cancer agent, another anti-inflammatory agent, etc.
  • the pharmaceutical composition can be suitable for intravenous, intramuscular, subcutaneous, parenteral, epidermal, and other routes of administration.
  • the active ingredient can be coated with a material or otherwise loaded in a material or structure to protect it from the action of acids and other natural conditions that may inactivate it.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • the composition of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.
  • H2 and H3 CDR hydrogen bonds include the carbonyl of T59 to K443 of EGFR domain III is highly solvent exposed.
  • H3 makes an additional hydrogen bond between the carbonyl of T103 and K465 of EGFR domain III which is also highly solvent exposed ( Figure 1B) .
  • Figure 1B The solvent exposed nature of the above interactions prompted the current inventors to hypothesize that substitution of amino acid residues in CDRs L2D50, L1Y32, H2T59 or H3T103 with an amino acid containing a shorter side chain would cause only minor affinity loss, and might cause insignificant disruptions to the antibody/antigen interaction interfaces.
  • mutant antibodies Alanine-scanning techniques were used to make mutants with alanine substitution to each identified amino acid.
  • the mutant antibodies were generated with each of the mutant heavy or light chain pairing with a wild type (of Panitumumab) or mutant light or heavy chain. 8 resulting mutant antibodies (with their VH and VL regions and corresponding SEQ ID NOs shown in the below table) with single or double mutations were subject to EGFR binding measurement.
  • mutant antibodies codon optimization and gene synthesis were performed. Specific full-length heavy chain and light chain DNA were each cloned into a separate pcDNA3 plasmid. HEK293 cell transient transfection of the paired plasmids and one-step Protein A purification was used to prepare enough proteins for testing. Antibodies made in this format expressed well with decent yield and could be purified in high purity with one step protein A purification process ( Figure 2a, b, c) .
  • ELISA assay was used to exam and compare the EGFR binding capabilities between the mutant antibodies and control wild type (wt) antibody.
  • HRP-labeled goat anti-human IgG Fc antibody (Sigma, A0170) in 40000 dilution as a detection agent and TMB for colorimetric reaction, the plates read at 450/650 nm for absorbance on Microplate Reader (Molecular Devices, SpectraMax 190) and data analysis was performed using a dose response curve format four parameters logistic model.
  • Fig. 3 showed that mutant antibodies with single point alanine substitution at H2T59 (BB0500-2f1) or L2D50 (BB0500-2f2) had a small impact on EC 50 of EGFR binding activities.
  • the mutant antibody with single point alanine substitution at H3T103 (BB0500-2g1) had a huge impact on its EGFR binding activity (EC 50 ⁇ 100 fold change) and mutation at L1Y32 (BB0500-2g2) caused complete loss of EGFR binding activity.
  • BB0500-2f with alanine substitutions at both H2T59 and L2D50 did seem to have an additive effect, with minimal impact on EC50 yet an even lower upper plateau.
  • double alanine substitutions on H3T103/L2D50 (BB0500-2gf) , or H2T59/L1Y32 (BB0500-2fg) behaved very similarly to that of single point mutation at either H3T103 or L1Y32 respectively (BB0500-2g1 or BB0500-2g2) , suggesting that alanine substitution at either H3T103 or L1Y32 had a dominant effect on the overall binding activities of the mutant antibodies.
  • double mutations at both H3T103 and L1Y32 showed a decent binding activity, similar to that of H2T59 and L2D50 double mutations (BB0500-2f) .
  • This drastically rescued binding activities with double mutations suggested that, in addition to loss of side chain target interactions, each single mutation might induce conformation changes of H2 or L2 loop.
  • the mutant H2 loop might collide with wt L2 loop and vice versa.
  • the mutant H2 loop and mutant L2 loop might fit cohesively and as a result, mutant antibody with this particular pair of double mutations largely retained the overall EGFR binding activity.
  • Measurament of EGFR binging affinity of the mutant antibodies and control (wt) antibody to human EGFR is performed on Octet RED96e (Pall FortéBio) .
  • Affinity tests are performed in SD buffer (0.05 %Tween-20, 0.1 %BSA in PBS buffer, pH 7.4) using streptavidin Capture (SAC) biosensors.
  • biotin-EGFR protein or buffer are dispensed into 96-well microtiter plates at a volume of 200 ⁇ L per well.
  • SAC biosensors (Pall FortéBio) are pre-wet with SD buffer to establish a baseline before protein immobilization.
  • the biotin-EGFR protein is immobilized onto the biosensors with a coupling height of 1.2 nm.
  • Binding association of the mutant antibodies or control (wt) antibody with test articles is monitored for 40 sec, and subsequent disassociation in SD buffer is monitored for 60 sec. Mutant and control antibodies binding is evaluated at the concentrations from 3.125-100 nM. Data are generated automatically by the Data Acquisition software, and data analysis is performed using FortéBio Data Analysis software.
  • CE-SDS of non-reducing ADC was also performed to evaluate percentage of non-covalently linked components in the ADC product, like free light chains (L) , free heavy chains (H) , half antibodies (HL) , intact antibodies (LHHL) and antibodies missing one or two light chains (HHL or HH) .
  • antibodies containing wt Panitumumab variable sequences and alanine substitution sequences were made in a format containing special hinge sequences with an extra H-H inter chain disulfide bond upstream of the indigenous H-H double disulfide bond.
  • the extra H-H disulfide bond was more vulnerable to reduction than the rest of the inter chain disulfide bonds.
  • the cysteine residues forming this disulfide bond became preferred sites for drug conjugation.
  • the ADCs made of these engineered antibodies were predominantly made of DAR2 ADC species (Figure 4a: BB0500-2f with denibulin; and Figure 4b: BB0500-2g with eribulin) .
  • ADCs made of two mutant antibodies (BB0500-2f-eribulin and BB0500-2g-eribulin) exerted similarly potent cytotoxicity activities to EGFR high-expressing cells (A431 cells) as that of ADC made of antibody containing wt Panitumumab sequences (BB0500-2d-eribulin) .
  • All three ADCs were more potent to this EGFR high-expressing cells than the ADC made of the low affinity anti-EGFR antibody Nimotuzumab (BB05D3-eribulin in the figure) .
  • the cytotoxic potency of the ADCs made from the two mutant antibodies were closer to that of nimotuzumab-ADC, while ADC made of antibody containing wt Panitumumab sequences was significantly more potent.

Abstract

An isolated antibody includes a heavy chain having a variable domain comprising the amino sequence of SEQ ID NO: 1, and a light chain having a variable domain comprising the amino sequence of SEQ ID NO: 4. Also provided is an isolated antibody including a heavy chain having a variable domain comprising the amino sequence of SEQ ID NO: 5, and a light chain having a variable domain comprising the amino sequence of SEQ ID NO: 6. ADCs of the antibodies are also provided.

Description

MODIFIED EGFR ANTIBODY WITH REDUCED AFFINITY Background
Antibodies are key immune molecules acting against foreign pathogens. The development of monoclonal antibody (mAb) technology resulted in widespread use of monoclonal antibodies in research, diagnosis and treatment of diseases. The therapeutic use of first-generation mAb (mostly monospecific, bivalent mAb) achieved success in the treatment of a variety of diseases, including cancer, autoimmune, and infectious diseases. However, many diseases, such as solid tumors, have been shown to be quite resistant to antibody-based therapies.
Antibody Drug Conjugates (ADCs) are mAbs chemically linked to active drugs, and therefore, have both the specific targeting of mAbs and the cancer-killing ability of cytotoxic drugs. The ability to select specific mAbs-drug combination and advances in the linking the mAbs and drugs provides new possibilities to target cancers while minimizing exposure of healthy tissue. By 2019, a total of seven ADCs have been approved by the FDA, including: ado-trastuzumab emtansine (Kadcyla TM) , brentuximab vedotin (Adcetris TM) , inotuzumab ozogamicin (Besponsa TM) , gemtuzumab ozogamicin (Mylotarg TM) , polatuzumab vedotin-piiq (Polivy TM) , Enfortumab vedotin (Padcev TM) , and Trastuzumab deruxtecan (Enhertu TM) . All of them involve conjugation of a cytotoxic drug with a bivalent mAb. In addition to the seven ADC drugs that have been approved for marketing, a large number of ADCs are currently under clinical development.
The epidermal growth factor receptor (EGFR, also known as HER1 or c-erbB-1) is a 170 kDa transmembrane glycoprotein and a member of the tyrosine kinase family of cell surface receptors. EGFR is abnormally activated in many epithelial tumors, including those in non-small cell lung cancer, breast cancer, colorectal cancer, head and neck cancers, and prostate cancer. Abnormal activation of EGFR can arise from overexpression of the receptor, gene amplification, activating mutations, overexpression of receptor ligands, and/or loss of regulators of EGFR activity.
Interruption of EGFR signaling, either by blocking EGFR binding sites on the extracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity, can inhibit the growth of EGFR-expressing tumors and improve the patient's condition. Anti‐EGFR antibodies exert antitumor effects by binding the receptor at the cell surface to interfere with ligand binding, which leads to the inhibition of its downstream signaling pathway.
Several ligand-blocking antibodies for EGFR, including cetuximab, nimotuzumab, panitumumab, and necitumumab, have been approved for the treatment of various types of cancers. Panitumumab is a clinically proven antibody that targeted EGFR potently and specifically. However, clinical response to Panitumumab is often accompanied by significant toxicities due to normal tissue expression. Also, while the extremely high affinity (KD=10 -11M) of Panitumumab made it a potent blocker of EGFR/EGF engagement, it made Panitumumab undesirable for ADC construction because of the further compounded side effects with the cytotoxic drug.
Summary of the Invention
In one aspect of the present disclosure, an isolated antibody is provided, which comprises (a1) a heavy chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 18, respectively, and (b1) a light chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23, respectively. Another isolated antibody is provided, which comprises: (b1) a heavy chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, and (b2) a light chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 23, respectively.
In one aspect of the present disclosure, an isolated antibody is provided, which comprises: (a) a heavy chain having a variable domain comprising the amino sequence of SEQ ID NO: 1, and a light chain having a variable domain comprising the amino sequence of SEQ ID NO: 4; or (b) a heavy chain having a variable domain comprising the amino sequence of SEQ ID NO: 5, and a light chain having a variable domain comprising the amino sequence of SEQ ID NO: 6.
In another aspect of the present disclosure, an isolated antibody is provided, which comprises: (a) a heavy chain comprising the amino sequence of SEQ ID NO: 7, and a light chain comprising the amino sequence of SEQ ID NO: 10; or (b) a heavy chain comprising the amino sequence of SEQ ID NO: 13, and a light chain comprising the amino sequence of SEQ ID NO: 10; or (c) a heavy chain comprising the amino sequence of SEQ ID NO: 11, and a light chain comprising the amino sequence of SEQ ID NO: 12.
The antibody of the present application can be monoclonal antibody. In some embodiments, the antibody of the present disclosure can be deemed as the antibody Panitumumab, which is the first fully human monoclonal antibody directed against the EGFR, being mutated at one amino acid on its heavy chain and one amino acid on its light chain. The heavy chain mutation can be T103A while the light chain mutation is Y32A, or the heavy chain mutation can be T59A while the light chain mutation is D50A.
In further aspects, a nucleic acid molecule encoding the antibody described herein, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector are also provided.
In yet a further aspect, the present disclosure provides an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof that comprises the antibody described herein conjugated to a cytotoxic drug molecule by a chemical linker. In some embodiments, the cytotoxic drug can be selected from the group consisting of monomethyl auristatin E (MMAE) , monomethyl auristatin F (MMAF) , auristatin E, auristatin F, maytansine DM1 and DM4, maytansinol, sandramycin, pyrrolobenzodiazepine dimer, anthracyclines, calicheamicin, dolastatin 10, duocarmycin, doxorubicin, thailanstatin A, uncialamycin, amanitins, ricin, diphtheria toxin, eribulin,  131I, interleukins, tumor necrosis factors, chemokines, and nanoparticles.
The chemical linker linking the antibody portion and the cytotoxic drug can be cleavable or non-cleavable. In some embodiments, the linker comprises a PEGn spacer where n is between 1 and 20 (i.e., having 1 to 20 repeat units (CH 2CH 2O) ) . In some embodiments, the chemical linker further comprises a linker segment connected to the PEGn spacer. In some embodiments, the chemical linker comprises a linker segment but does not comprise a PEGn spacer. In some embodiments, the linker segment can be selected from the group consisting of 6-maleimidocaproyl (MC) , maleimidopropionyl (MP) , valine-citrulline (val-cit) , alanine-phenylalanine (ala-phe) , p-aminobenzyloxycarbonyl (PAB) , 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB) , Val-Cit-PABC, Phe-Lys (Fmoc) -PAB, Aloc-D-Ala-Phe-Lys (Aloc) -PAB-PNP, Boc-Phe- (Alloc) Lys-PAB-PNP, and perfluorophenyl 3- (pyridine-2-yldisulfanyl) propanoate, or combinations thereof.
Brief Description of the Drawings
Figure 1 shows schematic diagrams of interactions between paratope surfaces of EGFR protein and CDRs of panitumumab. (A) Panitumumab light chain CDR interactions with the final β-strand of EGFR domain III. EGFR on top interactions with L1, L2 and L3 CDRs of the panitumumab on the bottom. (B) Panitumumab heavy chain CDR interactions with the β-sheet surface of EGFR domain III that binds EGFR. EGFR is shown on top with H1, H2, and H3 CDR regions, . Amino acids subject to mutation are circled.
Figure 2 shows characterization of antibodies according to some embodiments of the present invention. (a) purity and yields of certain antibodies produced in HEK293 cells; (b) SDS-PAGE plots of reducing (R) and Non-Reducing (NR) antibodies; (c) SEC-HPLC analysis of purified antibodies.
Figure 3 shows binding Curves of certain antibodies according to embodiments of the present invention, among other antibodies, to EGFR.
Figure 4 shows HIC profiles of two ADCs made of certain antibodies according to embodiments of the present invention. HIC profiles of two ADCs made of two different mutant antibodies.
Figure 5 shows cytotoxicity curves of certain ADCs according to embodiments of the present invention and comparative ADCs to EGFR-expressing cancer cells: (a) A431 cell, (b) NUGC3 cell.
Detailed Description
The present disclosure provides antibodies based on modification of the amino acid sequences of panitumumab. Without losing the binding specificity to EGFR, the modified (or mutant) antibody has reduced affinity to the antigen, and has reduced toxicities on normal tissues. ADCs based on these antibodies are also provided. According to some embodiments, compared with the ADC made from the parent wild-type panitumumab and a same drug, ADCs made of the mutant antibodies retain comparable potency in terms of killing EGFR high-expressing tumor cells, while having significantly reduced potencies to EGFR low-expressing cells. Therefore, treatment with an ADC made of such mutant antibody can have a better therapeutic window with less on-target-off-tumor toxicities to EGFR low-expressing normal skin tissues that was associated with Panitumumab treatment.
The terms “monoclonal antibody” as used herein refer to a preparation of antibody molecules of single molecular composition.
The complementarity-determining regions (CDRs) of an antibody are defined by those skilled in the art using a variety of methods/systems. These systems and/or definitions have been developed and refined over a number of years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and is commonly used. The Chothia definition is based on the location of the structural loop regions. The IMGT system is based on sequence variability and location within the structure of the variable domain. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analyses of the available antibody crystal structures. An Exemplary system is a combination of Kabat and Chothia.
In one aspect of the present disclosure, an isolated antibody is provided, which comprises (a1) a heaving chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 18, respectively, and (b1) a light chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23, respectively. Another isolated antibody is provided, which comprises: (b1) a heaving chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, and (b2) a light chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 23, respectively.
In one aspect of the present disclosure, an isolated antibody is provided, which comprises: (a) a heavy chain having a variable domain comprising the amino sequence of SEQ ID NO: 1, and a light chain having a variable domain comprising the amino sequence of SEQ ID NO: 4; or (b) a heavy chain having a variable domain comprising the amino sequence of SEQ ID NO: 5, and a light chain having a variable domain comprising the amino sequence of SEQ ID NO: 6. In another aspect of the present disclosure, an isolated antibody is provided, which comprises: (a) a heavy chain comprising the amino sequence of SEQ ID NO: 7, and a light chain comprising the amino sequence of SEQ ID NO: 10; or (b) a heavy chain comprising the amino sequence of SEQ ID NO: 13, and a light chain comprising the amino sequence of SEQ ID NO: 10; or (c) a heavy chain  comprising the amino sequence of SEQ ID NO: 11, and a light chain comprising the amino sequence of SEQ ID NO: 12.
DNA encoding an amino acid sequence variant of a starting polypeptide can prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the polypeptide. Variants of recombinant antibodies may be constructed also by restriction fragment manipulation or by overlap extension PCR with synthetic oligonucleotides. Mutagenic primers encode the cysteine codon replacement (s) . Standard mutagenesis techniques can be employed to generate DNA encoding such mutant engineered antibodies.
In yet a further aspect, the present disclosure provides a nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any of the antibody described herein. A host cell (e.g., a CHO cell, a human embryonic kidney cell, a lymphocytic cell, or microorganisms, such as E. coli, and fungi, such as yeast) containing an expression vector containing the nucleic acid molecule, can be used to produce antibodies of the present disclosure, preferably monoclonal antibodies. In one embodiment, DNA encoding partial or full-length antibody of the present disclosure can be obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operatively linked to transcriptional and translational regulatory sequences. The term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody genes. Such regulatory sequences are described, e.g., in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) ) . Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) , Simian Virus 40 (SV40) , adenovirus, e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, nonviral regulatory sequences can be used, such as the ubiquitin promoter or β-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRα promoter system, which contains sequences  from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8: 466-472) . The expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
The antibody encoding DNA can be inserted into the expression vector. The recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody encoding DNA can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody encoding DNA. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein) .
In a further aspect, the present disclosure provides an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof that comprises the antibody described herein conjugated to a cytotoxic drug molecule by a chemical linker. In some embodiments, the cytotoxic drug can be selected from the group consisting of monomethyl auristatin E (MMAE) , monomethyl auristatin F (MMAF) , auristatin E, auristatin F, maytansine DM1 and DM4, maytansinol, sandramycin, pyrrolobenzodiazepine dimer, anthracyclines, calicheamicin, dolastatin 10, duocarmycin, doxorubicin, thailanstatin A, uncialamycin, amanitins, ricin, diphtheria toxin, eribulin,  131I, interleukins, tumor necrosis factors, chemokines, and nanoparticles.
The chemical linker linking the antibody portion and the cytotoxic drug can be cleavable or non-cleavable. In some embodiments, the linker comprises a PEGn spacer where n is between 1 and 20 (i.e., having 1 to 20 repeat units (CH 2CH 2O) ) . In some embodiments, the chemical linker further comprises a linker segment connected to the PEGn spacer. In some embodiments, the chemical linker comprises a linker segment but does not comprise a PEGn spacer. In some embodiments, the linker segment can be selected from the group consisting of 6-maleimidocaproyl (MC) , maleimidopropionyl (MP) , valine-citrulline (val-cit) , alanine-phenylalanine (ala-phe) , p-aminobenzyloxycarbonyl (PAB) , 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB) , Val-Cit-PABC, Phe-Lys (Fmoc) -PAB, Aloc-D-Ala-Phe-Lys (Aloc) -PAB-PNP, Boc-Phe- (Alloc) Lys-PAB-PNP, and perfluorophenyl 3- (pyridine-2-yldisulfanyl) propanoate, or combinations thereof.
In further aspect, the present disclosure provides a pharmaceutical composition comprising one or more antibodies, ADCs or the pharmaceutically acceptable salts thereof, of the present invention, together with a pharmaceutically acceptable carrier. As used herein, “pharmaceutically  acceptable carrier” includes pharmaceutically acceptable carriers, excipients or stabilizers. These include but are not limited solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and the like that are physiologically compatible. The selection of suitable carrier is within the knowledge of an artisan skilled in the art. The composition may comprise one or more additional pharmaceutically active ingredients, such as another antibody, a drug, e.g., a cytotoxic or anti-tumor agent. The pharmaceutical compositions of the invention also can be administered in a combination therapy with, for example, another anti-cancer agent, another anti-inflammatory agent, etc.
The pharmaceutical composition can be suitable for intravenous, intramuscular, subcutaneous, parenteral, epidermal, and other routes of administration. Depending on the route of administration, the active ingredient can be coated with a material or otherwise loaded in a material or structure to protect it from the action of acids and other natural conditions that may inactivate it. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the composition of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.
Examples
1. Design of Panitumumab mutant antibodies with lower antigen affinities.
A high-resolution 3D structure analysis of Panitumumab/EGFR complex revealed that, while L3 CDR commonly make the largest contributions to antigen binding, the L2 CDR makes only a single interaction through a solvent exposed salt bridge between D50 and K465 of EGFR domain III, and the L1 CDR makes another single interaction through a solvent exposed hydrogen bond bridge between Y32 and I466 of EGFR domain III (Figure 1A) . This is consistent with the general  observation that L1/L2 CDR often makes minor binding contribution. On the heavy chain side, while H2 and H3 CDRs account for most of the central buried specific interactions between Panitumumab and EGFR domain III, a H2 CDR hydrogen bonds include the carbonyl of T59 to K443 of EGFR domain III is highly solvent exposed. H3 makes an additional hydrogen bond between the carbonyl of T103 and K465 of EGFR domain III which is also highly solvent exposed (Figure 1B) . The solvent exposed nature of the above interactions prompted the current inventors to hypothesize that substitution of amino acid residues in CDRs L2D50, L1Y32, H2T59 or H3T103 with an amino acid containing a shorter side chain would cause only minor affinity loss, and might cause insignificant disruptions to the antibody/antigen interaction interfaces.
Alanine-scanning techniques were used to make mutants with alanine substitution to each identified amino acid. The mutant antibodies were generated with each of the mutant heavy or light chain pairing with a wild type (of Panitumumab) or mutant light or heavy chain. 8 resulting mutant antibodies (with their VH and VL regions and corresponding SEQ ID NOs shown in the below table) with single or double mutations were subject to EGFR binding measurement.
Table 1: List of mutant antibodies
Figure PCTCN2020130417-appb-000001
For expression of the mutant antibodies, codon optimization and gene synthesis were performed. Specific full-length heavy chain and light chain DNA were each cloned into a separate pcDNA3 plasmid. HEK293 cell transient transfection of the paired plasmids and one-step Protein A purification was used to prepare enough proteins for testing. Antibodies made in this format expressed well with decent yield and could be purified in high purity with one step protein A purification process (Figure 2a, b, c) .
2. Measurement of EGFR binding affinities of the mutant antibodies
ELISA assay was used to exam and compare the EGFR binding capabilities between the mutant antibodies and control wild type (wt) antibody. Mutant antibody samples or control antibodies diluted in 5-fold serial dilutions starting from 10 μg/mL and 8 dilutions total, were coated onto 96-well plates and the plates were incubated at 4℃ overnight. Using HRP-labeled goat anti-human IgG Fc antibody (Sigma, A0170) in 40000 dilution as a detection agent and TMB for colorimetric reaction, the plates read at 450/650 nm for absorbance on Microplate Reader (Molecular Devices, SpectraMax 190) and data analysis was performed using a dose response curve format four parameters logistic model.
The results in Fig. 3 showed that mutant antibodies with single point alanine substitution at H2T59 (BB0500-2f1) or L2D50 (BB0500-2f2) had a small impact on EC 50 of EGFR binding activities. In contrast, the mutant antibody with single point alanine substitution at H3T103 (BB0500-2g1) had a huge impact on its EGFR binding activity (EC 50～100 fold change) and mutation at L1Y32 (BB0500-2g2) caused complete loss of EGFR binding activity. These results suggested that while H2T59 and L2D50 were minimally contributing to the overall target binding activity of Panitumumab, H3T103 and L1Y32 were either critically involved in the high-affinity association of Panitumumab/EGFR complex, or alanine substitutions at these places were detrimental to the proper conformation of the antigen binding surface. The upper plateau of the binding curves of neither BB0500-2f1 nor BB0500-2f2 reached to the level of wt control, suggesting that both substitutions impacted the off rate of the target binding kinetics.
Binding profiles of the double mutations revealed a much more complicated picture. First, BB0500-2f with alanine substitutions at both H2T59 and L2D50 did seem to have an additive effect, with minimal impact on EC50 yet an even lower upper plateau. Secondly, double alanine substitutions on H3T103/L2D50 (BB0500-2gf) , or H2T59/L1Y32 (BB0500-2fg) behaved very similarly to that of single point mutation at either H3T103 or L1Y32 respectively (BB0500-2g1 or BB0500-2g2) , suggesting that alanine substitution at either H3T103 or L1Y32 had a dominant effect on the overall binding activities of the mutant antibodies. Finally, and most surprisingly, double mutations at both H3T103 and L1Y32 (BB0500-2g) showed a decent binding activity, similar to that of H2T59 and L2D50 double mutations (BB0500-2f) . This drastically rescued binding activities with double mutations suggested that, in addition to loss of side chain target interactions, each single mutation might induce conformation changes of H2 or L2 loop. The  mutant H2 loop might collide with wt L2 loop and vice versa. In contrast the mutant H2 loop and mutant L2 loop might fit cohesively and as a result, mutant antibody with this particular pair of double mutations largely retained the overall EGFR binding activity.
Measurament of EGFR binging affinity of the mutant antibodies and control (wt) antibody to human EGFR is performed on Octet RED96e (Pall FortéBio) . Affinity tests are performed in SD buffer (0.05 %Tween-20, 0.1 %BSA in PBS buffer, pH 7.4) using streptavidin Capture (SAC) biosensors. biotin-EGFR protein or buffer are dispensed into 96-well microtiter plates at a volume of 200 μL per well. SAC biosensors (Pall FortéBio) are pre-wet with SD buffer to establish a baseline before protein immobilization. The biotin-EGFR protein is immobilized onto the biosensors with a coupling height of 1.2 nm. Binding association of the mutant antibodies or control (wt) antibody with test articles is monitored for 40 sec, and subsequent disassociation in SD buffer is monitored for 60 sec. Mutant and control antibodies binding is evaluated at the concentrations from 3.125-100 nM. Data are generated automatically by the Data Acquisition software, and data analysis is performed using FortéBio Data Analysis software.
The binding affinity data is presented in Table 2. Although the calculated K D of wt control antibody (Panitumumab) is higher than the published data (K D = 10 -11M) , the K D of the two double mutants (BB0500-2f and BB0500-2g) had over 100-fold reduction (in the range of 10 -10M) . The reductions were largely caused by faster dissociation rates with little changes in association rates. These data were in consistent with the ELISA data and support the hypothesis that the reduced saturation binding capacity of mutant antibodies was indeed cause by faster dissociation of the mutant antibody/target complex.
Table 2: Results of Kinetic Binding Affinity Parameters to Mutant and wt Antibodies
Figure PCTCN2020130417-appb-000002
3. Conjugation of mutant anti-EGFR antibodies to generate ADCs.
Under mild reduction conditions (TCEP : mAb = 1.1-2.4, PH7.2-7.4, 22-26℃ for <240min) , interchain disulfide bonds of an antibodies could be partially reduced and conjugated with drug- linker to form ADCs. To generated the drug conjugates, a purified antibody in phosphate buffer at neutral pH was added TCEP for partial reduction. Drug-linker (MC-val-cit-PAB-eribulin or MC-DM1) in DMA was added and allowed to react with antibody to obtain desired drug-to-antibody ratio (DAR) . To characterize the antibodies and ADCs, Hydrophobic Interaction Chromatography (HIC) was performed for the evaluation of drug distribution and molar ratio of drug and antibody in ADC. CE-SDS of non-reducing ADC was also performed to evaluate percentage of non-covalently linked components in the ADC product, like free light chains (L) , free heavy chains (H) , half antibodies (HL) , intact antibodies (LHHL) and antibodies missing one or two light chains (HHL or HH) .
To make antibodies suitable for site specific drug conjugation, antibodies containing wt Panitumumab variable sequences and alanine substitution sequences were made in a format containing special hinge sequences with an extra H-H inter chain disulfide bond upstream of the indigenous H-H double disulfide bond. The extra H-H disulfide bond was more vulnerable to reduction than the rest of the inter chain disulfide bonds. As a result, the cysteine residues forming this disulfide bond became preferred sites for drug conjugation. The ADCs made of these engineered antibodies were predominantly made of DAR2 ADC species (Figure 4a: BB0500-2f with denibulin; and Figure 4b: BB0500-2g with eribulin) .
4. Cytotoxicity of ADCs made of mutant antibodies to EGFR high/low-expressing cells.
To investigate the cytotoxicity of the mutant ADCs (BB0500-2f/2g-eribulin) , in vitro cytotoxicity to target expressing cancer cells with varying EGFR expression levels was evaluated in comparison with low affinity ADC (nimotuzumab-eribulin) and wild type Panitumumab ADC (Ctl-eribulin) in a colorimetric-based cytotoxic assay. To perform the assay, target cells were seeded into a 96-well flat-bottom tissue culture plate at an optimized cell density for each cell line and incubated at 37℃, 5 %CO 2 overnight (16-20 hrs) . Serial dilutions of ADC samples were transferred to cell plate and the assay plates were incubated for a defined period of time (3-5 days depend on cell lines) for optimal killing. Data analysis was performed using a dose response curve by four parameters logistic model. As shown in Figure 5A, ADCs made of two mutant antibodies (BB0500-2f-eribulin and BB0500-2g-eribulin) exerted similarly potent cytotoxicity activities to EGFR high-expressing cells (A431 cells) as that of ADC made of antibody containing wt Panitumumab sequences (BB0500-2d-eribulin) . All three ADCs were more potent to this EGFR  high-expressing cells than the ADC made of the low affinity anti-EGFR antibody Nimotuzumab (BB05D3-eribulin in the figure) . In contrast, to an EGFR low-expressing NUGC3 cell line, the cytotoxic potency of the ADCs made from the two mutant antibodies (BB0500-2f/2g) were closer to that of nimotuzumab-ADC, while ADC made of antibody containing wt Panitumumab sequences was significantly more potent.
While specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details according to all teachings that have been disclosed, and all of these changes fall within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Figure PCTCN2020130417-appb-000003
Figure PCTCN2020130417-appb-000004
Figure PCTCN2020130417-appb-000005

Claims (10)

  1. An isolated antibody comprising:
    (a1) a heavy chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 18, respectively, and (b1) a light chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23, respectively; or
    (a2) a heavy chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, and (b2) a light chain variable domain comprising a CDR1 region, a CDR2 region, and a CDR3 region comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 23, respectively.
  2. An isolated antibody comprising:
    (a) a heavy chain having a variable domain comprising the amino sequence of SEQ ID NO: 1, and a light chain having a variable domain comprising the amino sequence of SEQ ID NO: 4; or
    (b) a heavy chain having a variable domain comprising the amino sequence of SEQ ID NO: 5, and a light chain having a variable domain comprising the amino sequence of SEQ ID NO: 6.
  3. An isolated antibody comprising:
    (a) a heavy chain comprising the amino sequence of SEQ ID NO: 7, and a light chain comprising the amino sequence of SEQ ID NO: 10; or
    (b) a heavy chain comprising the amino sequence of SEQ ID NO: 13, and a light chain comprising the amino sequence of SEQ ID NO: 10; or
    (c) a heavy chain comprising the amino sequence of SEQ ID NO: 11, and a light chain comprising the amino sequence of SEQ ID NO: 12.
  4. The isolated antibody of any of claims 1-3, wherein the antibody is a monoclonal antibody.
  5. A nucleic acid molecule encoding the antibody of any one of claims 1-3.
  6. An expression vector containing the nucleic acid molecule of claim 5.
  7. A host cell containing the expression vector of claim 6.
  8. An antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, comprising: an antibody of any of the claims 1-3 conjugated to a cytotoxic drug by a chemical linker.
  9. The ADC or a pharmaceutically acceptable salt thereof, of claim 8, wherein the cytotoxic drug is selected from the group consisting of eribulin, monomethyl auristatin E (MMAE) , monomethyl auristatin F (MMAF) , auristatin E, auristatin F, maytansine DM1 and DM4, maytansinol, sandramycin, pyrrolobenzodiazepine dimer, anthracyclines, calicheamicin, dolastatin 10, duocarmycin, doxorubicin, thailanstatin A, uncialamycin, amanitins, ricin, diphtheria toxin,  131I, interleukins, tumor necrosis factors, chemokines, and nanoparticles.
  10. The ADC or a pharmaceutically acceptable salt thereof, of any of claims 8-9, wherein the chemical linker comprises a portion that is selected from the group consisting of 6-maleimidocaproyl (MC) , maleimidopropionyl (MP) , valine-citrulline (val-cit) , alanine-phenylalanine (ala-phe) , p-aminobenzyloxycarbonyl (PAB) , 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB) , Val-Cit-PABC, Phe-Lys (Fmoc) -PAB, Aloc-D-Ala-Phe-Lys (Aloc) -PAB-PNP, Boc-Phe- (Alloc) Lys-PAB-PNP, and perfluorophenyl 3- (pyridine-2-yldisulfanyl) propanoate.
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CN202180070808.5A CN116419929B (en) 2020-11-20 2021-11-19 Engineered EGFR antibodies with reduced affinity, drug conjugates and uses thereof
US18/253,681 US20240009318A1 (en) 2020-11-20 2021-11-19 Modified egfr antibody with reduced affinity, drug conjugate, and use thereof
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