WO2023212906A1 - Antibodis comprising engineered hinge and uses thereof - Google Patents
Antibodis comprising engineered hinge and uses thereof Download PDFInfo
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- WO2023212906A1 WO2023212906A1 PCT/CN2022/091130 CN2022091130W WO2023212906A1 WO 2023212906 A1 WO2023212906 A1 WO 2023212906A1 CN 2022091130 W CN2022091130 W CN 2022091130W WO 2023212906 A1 WO2023212906 A1 WO 2023212906A1
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- antibody
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Classifications
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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
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- 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
- A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
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- A—HUMAN NECESSITIES
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- 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
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- 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
- A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- 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
Abstract
Provided herein is an engineered dimeric antibody comprising in each monomer from N-terminal to C-terminal a Fab domain operably linked to an engineered hinge region and a Fc region, which is desirable for production of antibody drug conjugates.
Description
The present invention relates to the field of bio-pharmaceutical, and more particularly, engineered antibodies and antibody drug conjugates.
Antibody drug conjugate (ADC) is a novel targeting drug comprised by an antibody for targeting, a connector /linker for drug attachment and a high potent payload as effector. Antibody brings the cytotoxic drugs to antigen-expressing cells by antibody-antigen interaction. The targeting effect of the antibody makes the drug tumor-specific and less harmful to normal tissues. Thus, an otherwise very high-potent cytotoxin can be linked to an antibody to form an ADC for clinical use. An ADC enlarges the therapeutic window by reducing Minimum Effect Dose (MED) and elevating Maximum Tolerance Dose (MTD) .
A successful ADC development depends on multiple factors, including antibody selection, linker-payload selection, manner of linker-payload conjugation and conjugation process development. Cysteine thiols in antibody as strong nucleophiles are ideal reaction groups for conjugation. Since Cysteine residues exist as disulfide bonds in native form of antibodies, reduction of disulfide bonds between light-heavy chain and heavy-heavy chain (called inter-chain disulfides) in antibody provides perfect free Cysteine thiols for conjugation. Yet, if all cysteines are reduced and attached with drugs, the overly-modified antibody will be unstable in the circulation and have bad PK. It has been reported that ADCs with an average drug-to-antibody ratio (DAR) of 4 (DAR4) exhibit the best therapeutic index in vivo. But due to the close reduction potentials among the four inter-chain disulfide bonds, number of free thiols in IgG1 antibody after partial reduction shows normal distribution with large σ value. ADC produced by conventional method therefore is a mixture of different DAR species, which contains DAR0 to DAR8 products. As to average DAR4 ADCs, DAR4 species only comprises around 40%, while unwanted species comprise the majority of the product. Situation is even worse for IgG4 type of antibody, where higher percentages of antibodies with no free thiol and with 8 free thiols are found when average number of free thiols per antibody gets to four. As discussed above, ADCs with too high a drug loading exhibit less ideal PK and probably high toxicity; while those with low drug loading exhibit insufficient therapeutic efficacy.
Therefore, increasing homogeneity of ADC products (preferably homogeneity of DAR4) is a long-sought goal. Conjugations based on point mutation (e.g. Cysteine for Thiomab
TM or Unnatural amino acids) or based on short peptide tags for enzymes (e.g. mTG (Microbial transglutaminase) , Sortase or FGE (Formylglycine-generating enzyme) ) are the most commonly used technologies for site specific conjugation to get homogeneous ADCs. However, there are several challenges in application of these technologies. For instance, it is unknown as of which site is better for mutation or tagging.
There exists the persistent need for ADC products having increased homogeneity, a desirable DAR and more reliable and convenient way to produce same. And, in a related aspect, there exists the need for engineered antibodies that are desirable for ADCs and their production and that can be produced in a more convenient and productive way.
Summary of the Invention
The present invention provides an engineered antibody comprising an engineered hinge region, which is found desirable for production of ADCs with improved properties including homogeneity and a desirable DAR.
In a first aspect, provided herein is an engineered dimeric antibody comprising in each monomer from N-terminal to C-terminal a Fab domain operably linked to an engineered hinge region and a Fc region, wherein the engineered hinge region comprises a sequence as set forth by any one of SEQ ID NOs: 1 to 9.
In a second aspect, provided herein is a nucleic acid molecule or a combination of nucleic acid molecules encoding the engineered antibody of the invention.
In a further aspect, provided herein is an antibody drug conjugate comprising an engineered antibody according to the present invention.
In a further aspect, provided herein is a composition comprising or consisting of a mixture of antibody drug conjugates according to the present invention, wherein, at least or more than 80%, 85%, 90%or 95%of the antibody drug conjugates have a drug-to-antibody ratio being 4.
In a further aspect, provided herein is a method of preparing antibody drug conjugates, comprising a step of conjugating a partially reduced antibody of the invention with a linker-payload compound.
In a further aspect, provided herein is an antibody drug conjugate product obtained by the method of the invention, wherein, at least or more than 80%, 85%, 90%or 95%of the antibody drug conjugates in the product have a drug-to-antibody ratio being 4.
In a further aspect, provided herein is use of an engineered antibody comprising an engineered hinge region according to the present invention for production of ADCs.
In a further aspect, provided herein is a method of treating a disease, disorder or condition in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of the antibody drug conjugate according to the present invention. Also provided herein is an antibody drug conjugate according to the present invention for use in treatment of a disease, disorder or condition in a subject in need thereof.
The present invention provides various advantages. In particular, for the engineered antibody according to the present invention, since native immunoglobulin G hinge sequences are used and swapped at their natural structural positions, without introduction of any de novo amino acid sequence, the obtained engineered antibody will cause less immunogenicity in vivo. Furthermore, the engineered antibody of the invention allows to obtain highly homogeneous ADC products and to achieve the optimal four linker-payloads attachment under proper conjugation conditions, wherein ADCs with four linker-payloads occupy a high percentage of over 80%in the products.
For the antibody drug conjugates according to the present invention, production can be significantly simplified. In an embodiment, the production can be carried out in a simple (one-pot) conjugation procedure comprising, first, partial reduction using a mild reductant, and then, conjugation in the same buffer. No other enzymes are needed in conjugation reaction with antibodies in this invention. The ADC products according to the present invention advantageously have a high homogeneity, and the percentage of DAR4 species can increases to over 80%, over 85%or higher. Furthermore, the ADCs of the invention can exhibit excellent cytotoxicity to diseased cells, such as cells bearing a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA) .
Brief Description of the Figures
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Figure 1: A schematic depiction of engineered antibodies of the invention.
Figure 2: The DAR profiles of ADC products prepared using a native IgG1 antibody and a native IgG4 antibody respectively in a comparative example.
Figure 3: Structure of antibody 886-45 and the DAR profile of an ADC product prepared using the antibody in an example according to the invention.
Figure 4: Structure of antibody 886-50 and the DAR profile of an ADC product prepared using the antibody in an example according to the invention.
Nomenclature and Definition
As used herein, singular forms preceded by "a" , "an" and "the" include plural reference unless the context clearly dictates otherwise. As well, the terms "a" (or "an" ) , "one or more" and "at least one" can be used interchangeably herein.
As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%.
In the present disclosure, one or more features in one embodiment can be combined with any one or more features in another embodiment without departing from the spirit and concept of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skills in the fields to which this invention pertains. All publications and patents specifically mentioned herein are incorporated by reference for all purposes. All references cited in this specification are to be taken as indicative of the level of the skill in the art, which should not be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
As used herein, the term "antibody" refers to an immunoglobulin that specifically binds to an antigen, for which examples include monoclonal antibodies, polyclonal antibodies, multispecific antibodies and bispecific (bivalent) antibodies. Typically, as in the case of a dimeric antibody, the antibody comprises two heavy chains and two light chains, wherein each monomer is composed of the paired heavy chain and light chain. There are five major classes of antibodies, i.e., IgA, IgD, IgE, IgG and IgM, wherein some can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
As used herein, the terms "Fab" , "Fab domain" and "Fab arm" can be used interchangeably, which refer to the domain consisting of a light chain coupled with a heavy chain along the variable region and first constant region in an immunoglobulin (e.g., an antibody) . Typically, the Fab domain may comprise one or more inter-chain disulfide bonds. The Fab domain is responsible for various antigen binding activities.
As used herein, the term "Fc region" , also known as "Crystallizable Fragment" , refers to the fragment consisting of the second (CH2) and the subsequent constant regions of a heavy chain, or refers to the fragment consisting of a portion of the hinge region, the second (CH2) and the subsequent constant regions of a heavy chain. And, as used herein, the term "Fc domain" in context of a dimeric antibody refers to the portion of the coupled heavy chains along the Fc region of each. The Fc regions have various effector functions such as ADCC and CDC.
As used herein, the term "hinge" or "hinge region" can be used interchangeably, which refer to the region that connects the C-terminus of the CH1 to the N-terminus of the CH2 region of the heavy chain. A hinge region may have a length of about 12-62 amino acid residues. In human IgG1, the hinge region spans residues 216 to 230 and in human IgG4 from residues 219 to 230 according to EU numbering. As used herein, the term "hinge domain" in context of a dimeric antibody refers to the portion of the coupled heavy chains along the hinge region of each. Hinge region is a flexible linker between the Fab and the Fc of antibody. Length and flexibility of the hinge region varies extensively among the IgG subclasses, IgG1, IgG2, IgG3, and IgG4. Taking IgG1 and IgG4 which are most commonly used as therapeutic biologics for example, the hinge region of IgG1 comprises 15 amino acids (e.g., EPKSCDKTHTCPPCP (SEQ ID NO: 10) ) and is very flexible, while IgG4 has a shorter hinge with only 12 amino acids. Wild-type IgG1 and IgG4 differ by one amino acid in the core hinge region (226-229 by EU numbering) : Cys-Pro-Pro-Cys in IgG1 and Cys-Pro-Ser-Cys in IgG4. Natural IgG4 presents an equilibrium between inter-and intra-chain cysteine disulfide bonds at the core hinge region, resulting in observable heavy chain arm exchange and the presence of IgG4 half molecules post secretion. S228P mutation for IgG4 (e.g., ESKYGPPCPPCP (SEQ ID NO: 11) ) has been confirmed to markedly stabilize the covalent interaction between IgG4 heavy-chains by preventing natural arm exchange, thus widely applied in IgG4 antibody development and production. The S228P mutation results in a poly-proline helix (PPCPPCP) in the IgG4 hinge, which when combined with the shorter IgG4 hinge length, will further restrict its flexibility compared to the IgG1 hinge. The flexibility difference between different hinges has important implications for antibody bio-conjugation because the cysteine residues located in a flexible hinge fragment are considered more reactive than the ones located in a rigid hinge. There are experiment evidences that both S228P IgG4 inter-heavy-light-chain and inter-heavy-heavy-chain disulfide bond are weakly reactive.
As used herein, the term "specific binding" or "specifically bind" refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen. An engineered antibody provided herein may specifically bind to an antigen with a binding affinity (K
D) of ≤ 10
-6 M (e.g., ≤ 5x10
-7 M, ≤ 2x10
-7 M, ≤ 10
-7 M, ≤ 5x10
-8 M, ≤2x10
-8 M, ≤ 10
-8 M, ≤ 5x10
-9 M, ≤ 2x10
-9 M, ≤ 10
-9 M, or ≤ 10
-10 M) . K
D as used herein refers to the ratio of the dissociation rate to the association rate (k
off/k
on) .
The term "operably link" or "operably linked" refers to a juxtaposition, with or without a spacer or linker, of two or more biological sequences of interest in such a way that they are in a relationship permitting them to function in an intended manner. When used with respect to polypeptides, it is intended to mean that the polypeptide sequences are linked in such a way that permits the linked product to have the intended biological function. For example, an antibody variable region may be operably linked to a constant region so as to provide for a stable product with antigen-binding activity. The term may also be used with respect to polynucleotides. For one instance, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., promoter, enhancer, silencer sequence, etc. ) , it is intended to mean that the polynucleotide sequences are linked in such a way that permits regulated expression of the polypeptide from the polynucleotide.
As used herein, the term "subject" refers to a human or a non-human animal subject. Non-human animals may be mammals, such as primates. Examples of non-human animal subjects include but are not limited to domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, and bears. Preferably, the subject is a human. A "subject in need thereof" refers to a subject in need of diagnosis, prognosis, amelioration, prevention and/or treatment of a disease, disorder or condition.
1. Engineered Antibody
In one aspect of the disclosure, provided herein is an engineered dimeric antibody comprising in each monomer from N-terminal to C-terminal a Fab domain operably linked to an engineered hinge region and a Fc region, wherein the engineered hinge region comprises a sequence as set forth by any one of SEQ ID NOs: 1 to 9.
SEQ ID NO. | |
1 | EPKSCKSKYGPPCPPCP |
2 | EPKSCKKYGPPCPPCP |
3 | |
4 | |
5 | EPKSCKKTPPCPPCP |
6 | EPKSCSKTPPCPPCP |
7 | EPKSCSKKTPPCPPCP |
8 | EPKSCSKKGPPCPPCP |
9 | EPKSCSKYTPPCPPCP |
The dimeric antibody can be formed of two paired monomers, wherein each monomer comprises a heavy chain coupled with a light chain. The engineered antibody may have an IgG format. The dimer is formed of the monomers coupled by inter-chain bonding, including inter-chain bonds and/or interactions. Examples of such inter-chain bonding include but are not limited to disulfide bonds, hydrogen bonds, electrostatic interaction, salt bridges, hydrophobic-hydrophilic interaction and Knobs-into-Holes mechanism. Generally, the engineered antibody of the invention may have a structure as schematically depicted in Figure 1.
The engineered antibody of the invention is characterized in one aspect by the engineered hinge region of the invention. The engineered hinge region comprises a portion derived from an IgG1 hinge region and a portion derived from an IgG4 hinge region such that the engineered hinge domain comprises at least two inter-chain disulfide bonds. Surprisingly, this is found as can help increase homogeneity of the ADC product, such as an increased proportion of the DAR4 species. Meanwhile, residues surrounding the hinge cysteine residues are optimized according to the invention, which is surprisingly found as can further increase homogeneity of the ADC product, such as the proportion of DAR4. In some embodiments, the ratio of DAR4 in total ADC products can be higher than 80%, or even higher than 90%.
The engineered antibody of the invention is useful for bio-conjugation at cysteine residues released via reduction of disulfide bonds using mild reduction reagents. The engineered hinge domain of the invention alters reducibility of the disulfide bonds in the hinge domain, which leads to selective reduction of the disulfide bonds in the Fab domains, when the antibody is partially reduced using a mild reductant. This can advantageously provide a highly homogeneous ADC product comprising predominantly the conjugates having four linker-payloads mostly attached at the Fab domains. In some embodiments, the ratio of the DAR4 species in total conjugates can be higher than 80%, 90%or even higher.
Generally, engineered antibodies with the engineered hinge region provide various advantages in ADC production and the ADC product per se, such as a high homogeneity, a better controlled DAR, simplified production, desirable pharmacokinetic properties and/or pharmcodynamic properties.
In context of the present invention, the Fab domain may derive from any antibodies, especially the therapeutic antibodies. In some embodiments, the Fab domain derives from an antibody that specifically binds to a tumor antigen (TA) , such as a tumor specific antigen (TSA) and a tumor-associated antigen (TAA) . Examples of the tumor antigen include, but are not limited to, CD20, CD38, CD123, ROR1, ROR2, BCMA, PSMA, SSTR2, SSTR5, CD19, FLT3, CD33, PSCA, ADAM 17, CEA, Her2, EGFR, EGFR-vIII, CD30, FOLR1, GD-2, CA-IX, Trop-2, CD70, CD38, mesothelin, EphA2, CD22, CD79b, GPNMB, CD56, CD138, CD52, CD74, CD30, CD123, RON, and ERBB2. Examples of TA-specific antibodies include, but are not limited to, Trastuzumab, Rituximab, Cetuximab, Bevacizumab, Panitumumab, Alemtuzumab, Matuzuma, Gemtuzumab, Polatuzumab, Inotuzumab, etc. In some embodiments, the Fab domain is from a human IgG antibody, such as a Fab domain of IgG1 or IgG4 subclass, preferably IgG1 subclass.
The engineered antibody further comprises a Fc domain, preferably a human IgG Fc domain. In some embodiments, the Fc domain can be of IgG1 or IgG4 subclass. Apparently, the same applies to the Fc region in the context of a heavy chain.
In a further aspect of the disclosure, provided here is a nucleic acid molecule or a combination of nucleic acid molecules encoding the engineered antibody of the present invention. In some embodiments, the nucleic molecule or molecules may be provided in the form of one or more vectors, especially expression vectors. As can be well understood by an ordinarily skilled person in the art, nucleic acids encoding the heavy chain and the light chain of an antibody can be cloned into separate expression vectors and co-transferred into a host to recombinantly produce the antibody, and it is also possible to insert the coding sequences for both chains in one expression vector. Any expression vectors and hosts known in the field are useful for the present invention. Examples include but are not limited to plasmids, viral vectors, synthetic vectors, bacteria hosts, yeasts, insect cells and animal cells, such as CHO cells. In some embodiments, said nucleic acid molecule or combination of nucleic acid molecules, like vectors, may be provided in the form of a kit, which may optionally comprise instruction on using the nucleic acid molecule or molecules to recombinantly produce the antibody.
2. Antibody drug conjugate
2.1. Antibody drug conjugate
In one aspect of the disclosure, provided herein is an antibody drug conjugate comprising the engineered antibody of the invention conjugated to one or more drug molecules through a linker.
The drug (also known as "payload" ) useful in the present invention is not particularly limited. Drugs useful in the present invention include cytotoxic drugs, particularly those which are used for cancer therapy. Such drugs include, but are not limited to, DNA damaging agents, DNA binding agents, anti-metabolites, enzyme inhibitors such as thymidylate synthase inhibitors and topoisomerase inhibitors, tubulin inhibitors, and toxins (for example, toxins of a bacterial, fungal, plant or animal origin) . Specific examples include, for example, taxol, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes including taxol, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin, esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, hemiasterlins, maytansinoids (including DM1, DM2, DM3, DM4) , auristatins including monomethylauristatin E (MMAE) , monomethylauristatin F (MMAF) and monomethylauristatin D (MMAD) . In some embodiments, the drug is auristatins, e.g., MMAE. Drugs can be linked to the linker via any suitable methods known in the art. In some embodiments, the drug is provided for conjugation in the form of a linker-payload compound as an intermediate, like in the case of "MC-vc-PAB-MMAE" .
The drug used in the present invention can be bound to an antibody via a linker. Various linkers for ADCs are known in the art. Linkers useful in the present invention are not particularly limited, as long as it includes a moiety capable reacting with a thoil group rendered by an antibody and thereby linking to the antibody. Particularly useful in the present invention are amleimido or haloactyl functionalized linkers. Examples include, but are not limited to -MC-vc-PAB- ("MC" : Maleimide-caproyl; "-vc-" : the dipeptide of -Val-Cit-; "PAB" : para-aminobenzyl) , -MC-GGFG- ("-GGFG-" : the tetrapeptide of –Gly-Gly-Phe-Gly-) , -MC-vc-, -MC-and -SMCC- (succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate) . In some embodiments, the linker is -MC-vc-PAB-.
In some embodiments, the linker-payloads are connected to the cysteine residues provided by selected inter-chain disulfide bonds opened by reduction. In some embodiments, the conjugate of the invention may have a drug-to-antibody ratio (DAR) ranging from about 2 to about 8, preferably about 2 to about 6, and more preferably about 4. The ratio may refer to an average ratio in a population, such as an average of DAR4 for a population of ADCs. And, in a preferred embodiment, the conjugate comprises the drug molecules mostly attached at Fab domains, and in some cases, comprises all the four drug molecules attached at Fab domains.
2.2. Preparation of antibody drug conjugate
According to the invention, linker-payloads are conjugated at cysteine residues freed from disulfide bonds via reduction using a mild reductant. Particularly, the engineered hinge domain according to the invention alters reducibility of the disulfide bonds in the hinge domain, which leads to selective reduction of the disulfide bonds in the Fab domains, when the antibody is partially reduced using a mild reductant. This can advantageously provide a highly homogeneous product comprising predominantly the conjugates having four linker-payloads mostly attached at the Fab domains.
In one aspect of the invention, herein is provided a method of preparing the antibody drug conjugate of the invention. In brief, the method may involve conjugation between the partially reduced antibody and a linker-payload. Preferably, the conjugation is conducted in a reduction buffer with an organic solvent as additive to help dissolve the linker-payload. Specifically, the method may comprise a step of conjugating a partially reduced antibody of the invention with a linker-payload compound bearing a maleimido or haloacetyl moiety via Michael addition reaction. The partially reduced antibody may be produced by partially reducing an engineered antibody of the invention using a mild reductant. In some embodiments, the method may comprise:
partially reducing an engineered antibody of the invention using a mild reductant, and
conjugating the partially reduced antibody with a linker-payload compound bearing a maleimido or haloacetyl moiety via Michael addition reaction.
In some embodiments, the mild reductant is TCEP or DTT. In some embodiments, the reductant/antibody ratio is about 1 to 20, preferably 3 to 10, such as about 2 to 5, about 4 to 10 or about 9 to 15. In some embodiments, the partial reduction is conducted at pH of about 4.0 to 8.0, such as about 5 to 6, such as about 5.5. In some embodiments, the partial reduction is conducted for a period of about 0.5 to 24 hours (hr) , such as about 1 to 20 hours, about 0.5 to 5 hours, about 4 to 10 hours or 9 to 15 hours. In some embodiments, the partial reduction is conducted at a temperature of about 4 to 37 ℃, preferably 4 to 15 ℃, such as about 5 to 10℃, about 9 to 15 ℃, about 14 to 20 ℃, about 19 to 25 ℃ or about 24 to 30 ℃.
In some embodiments, the conjugation is carried out in a buffer of pH ranging from about 4.0 to 8.0, such as about pH 5 to 6, such as pH 5.5, optionally in presence of an organic additive (e.g., organic solvent or organic co-solvent) at an amount of about 0.0%to 20.0%by weight, such as about 5.0%to 15.0%or about 10.0%to 15.0%. In some embodiments, the drug/antibody ratio may be about 5 to 20, such as about 4 to 10, about 9 to 15 or about 14 to 19; the reaction temperature may be about 4 to 37 ℃, such as about 5 to 10℃, about 9 to 15 ℃, about 14 to 20 ℃, about 19 to 25 ℃ or about 24 to 30 ℃; and/or the time of reaction may be about 0.5 to 4 hours, such as about 1 to 3 hours.
In some embodiments, the method of the invention provides a product comprising or consisting of a mixture of antibody drug conjugates, wherein at least about 80%, at least about 85%, at least about 90%, or at least about 95%of the mixture have a DAR of 4. In some cases, the conjugates having a DAR of 4 have the four drug molecules all linked at the Fab domains.
3. Composition
In a further aspect, provided herein is a composition comprising or consisting of a mixture of antibody drug conjugates according to the invention, wherein at least about 80%, at least about 85%, at least about 90%, or at least about 95%of the mixture have a DAR of 4. In some cases, the conjugates having a DAR of 4 have the four drug molecules all linked at the Fab domains.
In a further aspect, provided herein is a pharmaceutical composition comprising the antibody drug conjugate or the mixture thereof as described above and a pharmaceutically acceptable excipient.
4. Treatment
The antibody drug conjugate of the invention can be used for treating a disease, disorder or condition in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of the antibody drug-conjugate. Also provided herein is an antibody drug conjugate according to the present invention for use in treatment of a disease, disorder or condition in a subject in need thereof. The disease to be treated may include but are not be limited to cancers, including solid tumors and hematopoietic malignancies, especially those positive for HER2, Trop2, EGFR and/or CD20. Examples of such cancers include but are not limited to breast cancers, ovarian cancer, endometrial cancer, prostate cancer, gastric cancers, pancreatic cancers, hepatic cancers, lung cancers (e.g., NSCLC) , gastric cancers, glioma, bladder cancer, colorectal cancers, lymphomas (e.g., non-Hodgkin’s lymphoma (NHL) ) and leukemia.
Examples
The following Examples are provided merely for illustration, with no intends to limit scope of the invention.
Abbreviation
DMA: N, N’-Dimethylacetamide
DTT: 1, 4-Dithiothreitol
TCEP: Tris (2-carboxyethyl) phosphine
MC: Maleimide-caproyl
PAB: para-aminobenzyl
MMAE: Monomethyl auristatin E
HIC: Hydrophobic interaction chromatography
HPLC: High performance liquid chromatography
RP-HPLC: Reverse phase-High performance liquid chromatography
SEC: Size exclusion chromatography
IC50: The half maximal inhibitory concentration
MED: Minimum Effect Dose
MTD: Maximum Tolerance Dose
MWCO: Molecular weight cut-off
DAR: Drug-to-antibody ratio
eq: Molar ratio of reductant/antibody or pay-load/antibody
General Method
Antibody Preparation
All antibody molecules described in this document were codon optimized for Cricetulus griseus, synthesized, cloned into proprietary production vectors, and then maxi-prepared from TOP10 E. coli cells following standard molecular biology procedures.
CHO K1 host cells were seeded at 2-4E5 cells/mL in CD CHO medium 72 hours before transfection. The host cells were counted for cell density using Vi-CELL, centrifuged at 290 g for 7 min and then resuspended in pre-warmed fresh CD CHO medium prior to transfection. The re-suspended host cells were incubated in a Kuhner shaker (36.5℃, 75%humidity, 6%CO
2, 120 rpm) before use.
A total 4 mg of plasmids encoding the antibody of interest were added into the re-suspended host cells, followed by 12 mg polyetherimide. The transfected cultures were incubated in a Kuhner shaker at 36.5℃, 75%humidity, 6%CO
2, 120 rpm for 4 hours. After proprietary supplements were added, the transfected cultures were then incubated in a Kuhner shaker at 31 ℃, 75%humidity, 6%CO
2, 120 rpm for 9-10 days.
On the harvest day, transfected cultures were clarified by primary centrifugation at 1,000 g for 10 min, and secondary centrifugation at 10,000 g for 40 min, followed by sterile filtration through 0.22 μm filter. The supernatants were purified by ProA chromatography. The ProA eluate were neutralized by adding 1-2%neutralization buffer (1 M Tris-HCl, pH 9.0) and formulated in 20 mM Histidine-Acetate buffer, pH 5.5.
All proteins were subjected to quality control tests before conjugation, including reducing and non-reducing SDS-PAGE, SEC-HPLC, endotoxin level detection by LAL gel clot assay and molecular identification by mass spectrometry.
HIC-HPLC for DAR determination
Procedure: 8μl ADC samples were subjected to HIC-HPLC for DAR determination.
SEC-HPLC for purity and aggregation
Procedure: 20μl ADC samples were subjected to SEC-HPLC for purity and aggregation.
RP-HPLC for drug loading
Procedure: mix 20μl of ADC sample with 75μl 8M Guanidine-HCl and 5μl Tris-HCl, pH 8.0. Then add 1μl 0.5M TCEP solution into the mixture. The reaction was performed at 37 ℃ for 30min and then tested with RP-HPLC for drug loading on antibody.
RP-HPLC for free drug determination
Procedure: 85μl ADC solution was mixed with 15μl DMA and then protein was precipitated with 100μl precipitation buffer (37.5%v/v of Methanol in Acetonitrile, saturated with NaCl) and vortex at 1400rpm for 10min at 22 ℃.
Sample was centrifuged at 16000rpf for 10min. The supernatant was taken for RP-HPLC assay together with standard sample for free drug determination.
Cytotoxicity test
Tumor model cells (e.g., HCC1954 cells, Raji cells and HCC827 cells) in medium (e.g., RPMI-1640 medium supplemented with 10%fetal bovine serum) are plated (e.g., at a density of 4000 cells/well for HCC1954 cells, 10000 cells/well for Raji cells and 3000 cells/well for HCC827 cells) . The cells are treated with test ADCs, such as ADCs targeting Her2, CD20 and EGFR. For example, Raji cells can be treated with ADCs upon completion of cells plating, and HCC1954 and HCC827 cells can be treated with ADCs 24hr after the cells are plated. Results of treatment are then evaluated. For example, viability can be determined after 4 or 5 days of treatment with ADCs at 37℃, whereby maximum inhibition and IC
50 can be calculated.
Comparative Example 1
A native IgG1 antibody and a native IgG4 antibody without subclass swapping in domains were prepared as described above. The native IgG1 antibody had a hinge sequence of EPKSCDKTHTCPPCP (SEQ ID NO: 10) and the native IgG4 antibody a hinge sequence of ESKYGPPCPPCP (SEQ ID NO: 11) . The antibodies were dissolved in 50mM PB, 50mM NaCl, 2mM EDTA, PH 7.0 to a concentration of 8.0 mg/ml, 50mM PB and 50mM NaCl, 2mM EDTA, PH 6.5 to a concentration of 8.0 mg/ml, respectively. For the IgG1 antibody, 2.7 eq of TCEP was added and the mixture was incubated at 37 ℃ for 2hrs. For the IgG4 antibody, 4.1 eq of TCEP was added and the mixture was incubated at 37 ℃ for 24hrs.
Then, for each mixture, DMA was added to the reduced antibody to a concentration of 10%, followed by 7 eq (for IgG1) and 9 eq (for IgG4) of MC-vc-PAB-MMAE, respectively. Conjugation reaction was performed at 4 ℃ for 1hr. The conjugated products were purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. Final products were characterized with HIC-HPLC for DAR and drug distribution determination (Figure 2) .
Example 1
Antibodies 886-45 (IgG1-Fab, IgG1-Fc; Hinge sequence: EPKSCKSKTPPCPPCP (SEQ ID NO: 4) ; Heavy chain (HC) sequence: SEQ ID NO: 16, Light chain (LC) Sequence: SEQ ID NO: 12) was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 4.0mg/ml. 2.6eq of TCEP was added to the antibody solution and the mixture was incubated at 4 ℃ for 3hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 7eq of MC-vc-PAB-MMAE. Conjugation reaction was performed at 4 ℃ for another 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. Final products were characterized with HIC-HPLC for DAR and drug distribution determination (Figure 3) . With the engineering of hinge region, the ADC product was highly homogeneous, comprising predominantly the DAR4 species at a percentage as high as 80.20%. As seen, the engineering of hinge region according to the invention significantly increased homogeneity of ADC products, as compared with the native antibodies.
Example 2
Antibodies 886-50 (IgG1-Fab, IgG1-Fc; Hinge sequence: EPKSCSKYTPPCPPCP (SEQ ID NO: 9) ; HC sequence: SEQ ID NO: 21, LC Sequence: SEQ ID NO: 12) was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 4.0mg/ml. 3.1eq of TCEP was added to the antibody solution and the mixture was incubated at 4 ℃ for 3hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 7eq of MC-vc-PAB-MMAE. Conjugation reaction was performed at 4 ℃ for another 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. Final products were characterized with HIC-HPLC for DAR and drug distribution determination (Figure 4) . With the engineering of hinge region, the ADC product was highly homogeneous, predominantly comprising the DAR4 species at a percentage as high as 87.96%.
Example 3
Antibodies 886-42 (IgG1-Fab, IgG1-Fc; Hinge sequence: EPKSCKSKYGPPCPPCP (SEQ ID NO: 1) ; HC sequence: SEQ ID NO: 13, LC Sequence: SEQ ID NO: 12) was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 4.0mg/ml. 2.6eq of TCEP was added to the antibody solution and the mixture was incubated at 4 ℃ for 3hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 7eq of MC-vc-PAB-MMAE. Conjugation reaction was performed at 4 ℃ for another 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. Final products were characterized with HIC-HPLC for DAR and drug distribution determination. With the engineering of hinge region, the ADC product was highly homogeneous, predominantly comprising the DAR4 species at a percentage as high as 79.49%.
Example 4
Antibodies 886-43 (IgG1-Fab, IgG1-Fc; Hinge sequence: EPKSCKKYGPPCPPCP (SEQ ID NO: 2) ; HC sequence: SEQ ID NO: 14, LC Sequence: SEQ ID NO: 12) was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 4.0mg/ml. 2.2eq of TCEP was added to the antibody solution and the mixture was incubated at 4 ℃ for 3hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 7eq of MC-vc-PAB-MMAE. Conjugation reaction was performed at 4 ℃ for another 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. Final products were characterized with HIC-HPLC for DAR and drug distribution determination. With the engineering of hinge region, the ADC product was highly homogeneous, predominantly comprising the DAR4 species at a percentage as high as 76.35%.
Example 5
Antibodies 886-44 (IgG1-Fab, IgG1-Fc; Hinge sequence: EPKSCKTPPCPPCP (SEQ ID NO: 3) ; HC sequence: SEQ ID NO: 15, LC Sequence: SEQ ID NO: 12) was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 4.0mg/ml. 4.2eq of TCEP was added to the antibody solution and the mixture was incubated at 4 ℃ for 3hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 7eq of MC-vc-PAB-MMAE. Conjugation reaction was performed at 4 ℃ for another 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. Final products were characterized with HIC-HPLC for DAR and drug distribution determination. With the engineering of hinge region, the ADC product was highly homogeneous, predominantly comprising the DAR4 species at a percentage as high as 71.51%.
Example 6
Antibodies 886-46 (IgG1-Fab, IgG1-Fc; Hinge sequence: EPKSCKKTPPCPPCP (SEQ ID NO: 5) ; HC sequence: SEQ ID NO: 17, LC Sequence: SEQ ID NO: 12) was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 4.0mg/ml. 2.5eq of TCEP was added to the antibody solution and the mixture was incubated at 4 ℃ for 3hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 7eq of MC-vc-PAB-MMAE. Conjugation reaction was performed at 4 ℃ for another 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. Final products were characterized with HIC-HPLC for DAR and drug distribution determination. With the engineering of hinge region, the ADC product was highly homogeneous, predominantly comprising the DAR4 species at a percentage as high as 79.60%.
Example 7
Antibodies 886-47 (IgG1-Fab, IgG1-Fc; Hinge sequence: EPKSCSKTPPCPPCP (SEQ ID NO: 6) ; HC sequence: SEQ ID NO: 18, LC Sequence: SEQ ID NO: 12) was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 4.0mg/ml. 3.2eq of TCEP was added to the antibody solution and the mixture was incubated at 4 ℃ for 3hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 7eq of MC-vc-PAB-MMAE. Conjugation reaction was performed at 4 ℃ for another 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. Final products were characterized with HIC-HPLC for DAR and drug distribution determination. With the engineering of hinge region, the ADC product was highly homogeneous, predominantly comprising the DAR4 species at a percentage as high as 81.50%.
Example 8
Antibodies 886-48 (IgG1-Fab; IgG1-Fc, Hinge sequence: EPKSCSKKTPPCPPCP (SEQ ID NO: 7) ; HC sequence: SEQ ID NO: 19, LC Sequence: SEQ ID NO: 12) was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 4.0mg/ml. 2.5eq of TCEP was added to the antibody solution and the mixture was incubated at 4 ℃ for 3hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 7eq of MC-vc-PAB-MMAE. Conjugation reaction was performed at 4 ℃ for another 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. Final products were characterized with HIC-HPLC for DAR and drug distribution determination. With the engineering of hinge region, the ADC product was highly homogeneous, predominantly comprising the DAR4 species at a percentage as high as 82.44%.
Example 9
Antibodies 886-49 (IgG1-Fab, IgG1-Fc; Hinge sequence: EPKSCSKKGPPCPPCP (SEQ ID NO: 8) ; HC sequence: SEQ ID NO: 20, LC Sequence: SEQ ID NO: 12) was dissolved in 20mM Histidine-acetate pH 5.5 to a concentration of 4.0mg/ml. 2.5eq of TCEP was added to the antibody solution and the mixture was incubated at 4 ℃ for 3hr. Then DMA was added to the reduced antibody to a concentration of 10%, followed by 7eq of MC-vc-PAB-MMAE. Conjugation reaction was performed at 4 ℃ for another 1hr. The conjugated product was purified with 40KD MWCO desalting column and stored in 20mM Histidine-acetate pH 5.5. Final products were characterized with HIC-HPLC for DAR and drug distribution determination. With the engineering of hinge region, the ADC product was highly homogeneous, predominantly comprising the DAR4 species at a percentage as high as 81.20%.
Claims (14)
- An engineered dimeric antibody comprising in each monomer from N-terminal to C-terminal a Fab domain operably linked to an engineered hinge region and a Fc region, wherein the engineered hinge region comprises a sequence as set forth by any one of SEQ ID NOs: 1 to 9.
- The engineered antibody of claim 1, wherein the Fab domain is of IgG1 or IgG4 subclass, preferably IgG1 subclass.
- The engineered antibody of claim 1, wherein the Fc region is of IgG1 or IgG4 subclass.
- A nucleic acid molecule or a combination of nucleic acid molecules encoding the engineered antibody of claim 1.
- The nucleic acid molecule or combination of nucleic acid molecules of claim 4, wherein the nucleic acid molecule or each nucleic acid molecule of the combination is a vector.
- An antibody drug conjugate comprising an engineered antibody according to any one of claims 1-3.
- A composition comprising or consisting of a mixture of antibody drug conjugates according to claim 6, wherein, at least or more than 80%, 85%, 90%or 95%of the antibody drug conjugates have a drug-to-antibody ratio being 4.
- A method of preparing antibody drug conjugates, comprising a step of conjugating a partially reduced antibody of claim 1 with a linker-payload compound.
- The method of claim 8, further comprising a step of partially reducing an engineered antibody of claim 1 using a mild reductant to provide the partially reduced antibody.
- The method of claim 9, characterized in one or more the followings:wherein, the mild reductant is TCEP or DTT;wherein, the reductant/antibody ratio is 1 to 20, preferably 3 to 10;wherein, the step of partially reducing is conducted at pH of 4.0 to 8.0, preferably 5 to 6;wherein, the step of partially reducing is conducted for a period of 0.5 to 24 hours, preferably 1 to 20 hours; and/orwherein, the step of partially reducing is conducted at a temperature of 4 to 37 ℃, preferably 4 to 15 ℃.
- An antibody drug conjugate product obtained by the method of claim 8, wherein, at least or more than 80%, 85%, 90%or 95%of the antibody drug conjugates in the product have a drug-to-antibody ratio being 4.
- Use of an engineered antibody of claim 1 for production of ADCs.
- A method of treating a disease in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of the antibody drug conjugate according to claim 6.
- An antibody drug conjugate according to claim 6 for use in treatment of a disease in a subject in need thereof.
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2009006520A1 (en) * | 2007-07-03 | 2009-01-08 | Medimmune, Llc | Hinge domain engineering |
CN109071674A (en) * | 2015-10-30 | 2018-12-21 | 豪夫迈·罗氏有限公司 | The antibody fragment and preparation method of hinge modification |
CN114127117A (en) * | 2019-07-19 | 2022-03-01 | 上海药明生物技术有限公司 | Polypeptide complex for conjugation and uses thereof |
WO2022152308A1 (en) * | 2021-01-18 | 2022-07-21 | Wuxi Biologics (Shanghai) Co., Ltd. | Engineered anti-trop2 antibody and antibody-drug conjugate thereof |
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Publication number | Priority date | Publication date | Assignee | Title |
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WO2009006520A1 (en) * | 2007-07-03 | 2009-01-08 | Medimmune, Llc | Hinge domain engineering |
US20110077383A1 (en) * | 2007-07-03 | 2011-03-31 | Medimmune, Llc | Hinge domain engineering |
CN109071674A (en) * | 2015-10-30 | 2018-12-21 | 豪夫迈·罗氏有限公司 | The antibody fragment and preparation method of hinge modification |
CN114127117A (en) * | 2019-07-19 | 2022-03-01 | 上海药明生物技术有限公司 | Polypeptide complex for conjugation and uses thereof |
WO2022152308A1 (en) * | 2021-01-18 | 2022-07-21 | Wuxi Biologics (Shanghai) Co., Ltd. | Engineered anti-trop2 antibody and antibody-drug conjugate thereof |
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