Bifunctional degraders of igg4 immunoglobulins

Abstract

A composition of matter comprising: an IgG4 binding moiety, a cell receptor binding moiety that binds to a hepatocyte or other degrading cell through an asialoglycoprotein receptor (ASGPR) on the surface of the hepatocyte or other degrading cell, and optionally a linker moiety connecting the IgG4 binding moiety and the cell receptor binding moiety, wherein the composition of matter is useful for removing IgG4 from a subject or patient.

Description

Technical Field

[0001] This invention generally relates to a pharmaceutical formulation characterized by the use of an inactive ingredient, such as a carrier or inert additive, a targeting agent or modifier chemically bound to an active ingredient, or an inactive ingredient chemically bound to an active ingredient, such as a polymer-drug conjugate, wherein the inactive ingredient is a modifier, which is an antibody, an immunoglobulin, or a fragment thereof, such as an Fc fragment, wherein the modifier is an antibody or an immunoglobulin having at least one antigen-binding site, the antibody targeting a receptor, a cell surface antigen, or a cell surface determinant, and particularly relates to a bifunctional molecule for treating IgG4-related diseases having a circulating protein binding moiety linked to a cell receptor-binding moiety via a linker group. Background Technology

[0002] IgG4-related disease (IgG4-RD), formerly known as IgG4-related systemic disease, is a chronic inflammatory condition characterized by tissue infiltration of lymphocytes and IgG4-secreting plasma cells. In most people with this disease, serum IgG4 concentrations are elevated during the acute phase. Kamisawa et al., The Lancet, 385 (9976), 1460–1471 (April 11, 2015).

[0003] An international expert panel developed recommendations for the management of IgG4-related disease. Khosroshahi et al., Arthritis & Rheumatology, 67(7), 1688–1699 (July 2015); Walsh, MedPage Today (April 1, 2015). The panel concluded that all cases of symptomatic, active IgG4-related disease require treatment. Treatment was recommended for some organ manifestations, such as aortitis, retroperitoneal fibrosis, proximal biliary stricture, tubulointerstitial nephritis, dural meningitis, pancreatic enlargement, and pericarditis. Some cases of asymptomatic IgG4-related disease also require treatment, as some organs often do not develop symptoms until late in the disease.

[0004] Unless contraindicated, glucocorticoids are the recommended first-line agents for inducing remission, but this treatment does not address the underlying cause of the disease. The goal of glucocorticoid therapy is to induce and maintain remission to prevent the progression of fibrosis and organ destruction in affected organs. Relapse is common during or after glucocorticoid tapering. Therefore, the biomedical field still needs new drugs capable of treating IgG4-related diseases or slowing their progression. Summary of the Invention

[0005] This invention relates to a bifunctional composition (pharmaceutical) capable of binding to, removing from the bloodstream, and degrading IgG4 immunoglobulin.

[0006] In one embodiment, the present invention provides a substance composition (pharmaceutical) comprising:

[0007] IgG4 antibody binding site,

[0008] The cell receptor binding portion can bind to hepatocytes or other degrading cells via the desialylate glycoprotein receptor (ASGPR) on the surface of hepatocytes of patients or subjects, or via receptors on the surface of degrading cells.

[0009] The linker portion connects the IgG4 binding portion and the cell receptor binding portion, wherein the linker portion can be a peptide bond or a larger linker portion.

[0010] In some embodiments, the substance composition (pharmaceutical) has the following structure:

[0011]

[0012] [CPBM] is the IgG4 antibody binding portion;

[0013] [CRBM] is the cell receptor binding moiety;

[0014] Each [CON] is an optional connector chemical part, which, when present, connects directly to [CPBM] or [CRBM] or connects [LINKER] to [CPBM] or [CRBM], and

[0015] [LINKER] is a chemical moiety covalently attached to one or more [CRBM] or [CPBM] groups, or

[0016] Its pharmaceutically acceptable salts, stereoisomers, solvates, or polymorphs.

[0017] In some embodiments, the substance composition (pharmaceutical) has the following structure:

[0018] R CN −(Xaa)y−R CC ,

[0019]

[0020] [AGN101],

[0021]

[0022] [AGN102],

[0023]

[0024] [AGN103],

[0025]

[0026] [AGN104], or its salt.

[0027] In some specific and non-limiting embodiments, the material composition is selected from the group in Table 1.

[0028]

[0029] AGN402 features a lysine-based conjugation. In the IgG4 binding moiety, two identical single chains are covalently linked by a disulfide bond (ABT310, Fc fusion). To prepare the material composition, TBT105 is conjugated with a lysine amine in the nanobody fusion. During the synthesis of AGN402, the leaving group is eliminated when the payload is conjugated with the amine.

[0030] AGN508 has a cysteine-based conjugation. The IgG4 binding moiety has a single chain (ABT501, a nanobody). To prepare the composition, TBT103 containing maleimide is conjugated to the C-terminal cysteine ​​of ABT501 via a Michael addition reaction.

[0031] AGN501 has a cysteine-based conjugation. The IgG4 binding moiety has a single chain (ABT502, a nanobody). To prepare the composition, TBT103 containing maleimide is conjugated to the C-terminal cysteine ​​of ABT502 via a Michael addition reaction.

[0032] AGN502 has a cysteine-based conjugation. The IgG4 binding moiety has a single chain (ABT503, a nanobody). To prepare the composition, TBT103 containing maleimide is conjugated to the C-terminal cysteine ​​of ABT503 via a Michael addition reaction.

[0033] AGN509 has a cysteine-based conjugation. The IgG4 binding moiety has a single chain (ABT504, a nanobody). To prepare the composition, TBT103 containing maleimide is conjugated to the C-terminal cysteine ​​of ABT504 via a Michael addition reaction.

[0034] AGN401 has a cysteine-based conjugation. The IgG4 binding moiety has a single chain (ABT306, a nanobody). To prepare the composition, TBT103 containing maleimide is conjugated to the C-terminal cysteine ​​of ABT306 via a Michael addition reaction.

[0035] AGN403 has a conjugation based on sorting enzyme linkage. The IgG4 binding moiety has a single chain (ABT304). To prepare the composition, pentaglycine-modified TBT106 is conjugated to the C-terminal pentapeptide motif LPETG of ABT304 via sorting enzyme-catalyzed linkage.

[0036] AGN404 has a conjugation based on sorting enzyme linkage. The IgG4 binding moiety has a single chain (ABT305). To prepare the composition, pentaglycine-modified TBT106 is conjugated to the C-terminal pentapeptide motif LPETG of ABT305 via sorting enzyme-catalyzed linkage.

[0037] In another embodiment, the IgG4 antibody-binding portion is an antibody and an antibody-derived binding protein. In a particular, non-limiting embodiment, the complementarity-determining region of the IgG4 antibody-binding portion includes regions having the structures of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 according to the Chothia numbering scheme.

[0038] In another embodiment, the IgG4 antibody binding portion is a VHH nanobody.

[0039] In some specific and non-limiting embodiments, the IgG4 binding portion is selected from the group in Table 2.

[0040]

[0041] In another embodiment, the cell receptor binding portion comprises an ASGPR binding group according to the following chemical structure:

[0042]

[0043] [TBT101], or

[0044]

[0045] [TBT102],

[0046] The cell receptor binding portion contains additional elements described in this specification.

[0047] In some specific and non-limiting embodiments, the cell receptor binding portion is selected from the group in Table 3.

[0048]

[0049] In another embodiment, the present invention provides a composition of substances (pharmaceuticals) that is used as a drug.

[0050] In another embodiment, the present invention provides a composition (pharmaceutical) of substances (medications) for treating or preventing disease states or symptoms associated with IgG4 upregulation in patients or subjects.

[0051] In one aspect, the administered drug composition binds to IgG4 antibodies, particularly but not limited to IgG4 autoantibodies, in the circulation of the subject or patient to form a drug-antibody complex. This drug-antibody complex is then endocytosed by cells, particularly but not limited to hepatocytes, and released from ASGPR via calcium depletion in the endosome and protonation changes at the binding site due to pH reduction. The endocytosed proteins are transported to the endosome, which then fuses with lysosomes. Lysosomal proteases then degrade the endocytosed proteins, permanently removing them from circulation.

[0052] In another embodiment, the present invention provides a pharmaceutical composition comprising a substance composition and at least one pharmaceutically acceptable excipient.

[0053] In another embodiment, the present invention provides a method for preparing a material composition, wherein the method includes a conjugation step, wherein the conjugation step results in the attachment of an IgG4 binding moiety to a cell receptor binding moiety. In another embodiment, the method for preparing the material composition includes a conjugation step selected from the Markush group, which includes a MATE conjugation step, a maleimide conjugation step, and a sorting enzyme conjugation step.

[0054] In another embodiment, the present invention provides a method for preparing a molecule in which the IgG4 binding portion is linked to another molecule or device, wherein the method includes a conjugation step selected from the Markush group, which includes a MATE conjugation step, a maleimide conjugation step, a sorting enzyme conjugation step, and an AviTag™ conjugation step.

[0055] In another embodiment, the present invention provides a method for removing IgG4 from a patient or subject by administering an effective amount of a drug to the patient or subject.

[0056] In another embodiment, the present invention provides a method for treating or preventing a disease state or symptom associated with IgG4 upregulation in a patient or subject by administering a therapeutically effective amount of an agent to the patient or subject. The disease may be selected from the Markush group, which comprises pemphigus vulgaris, pemphigus foliaceus, anti-LGI1 encephalitis, and MuSK myasthenia gravis.

[0057] In another embodiment, the present invention provides a composition comprising an agent as a first compound and a second compound (at least one additional agent), the second compound comprising a portion capable of conjugating to an IgG4 binding portion of the first compound to form the first compound.

[0058] Several objects, features, aspects, and advantages of the present invention will become more apparent from the following detailed description of embodiments of the invention and the accompanying drawings. Attached Figure Description

[0059] For illustrative purposes, some embodiments of the invention are shown in the accompanying drawings described below. Similar reference numerals in the drawings consistently indicate similar elements. The invention is not limited to the precise arrangements, dimensions, and instruments shown.

[0060] Figure 1 The structure of one embodiment of a bifunctional substance composition (pharmaceutical) is shown.

[0061] Figure 2 This is a sequence comparison of the constant heavy chains of human IgG1 (SEQ ID NO: 2), IgG2 (SEQ ID NO: 3), and IgG4 (SEQ ID NO: 4). Sequence differences between IgG2 and IgG4 and IgG1 are indicated. Hinge regions and lower hinge regions, as well as the N-glycosylation site N297, are indicated. Numbering follows the EU numbering scheme. Sequences important for FcgR binding, C1q, and FcRn are shaded. Some mutants that enhance ADCC are shown above or below the sequences, such as: Xencor's S239D, A330L, and I332E; Genentech's S298A, E333A, and K334A; and Eli Lilly / AME's P247I and A339D / Q. Examples of FcRn-binding mutants with extended half-life are also shown, as described: MedImmune's YTE mutants (M252Y, S254T, T256E), PDL's T250Q and M428L mutants, Sally Ward's H433K and N434Y mutants, and Genentech's N434A mutant are shown in the boxes.

[0062] Figure 3 This is a table of the R groups of the MATE reagent and the bifunctional MoDE final compound (Table 4).

[0063] Figure 4 This is a set of schematic diagrams of four of the material composition examples, particularly AGT401, AGT402, AGT403 and AGT404.

[0064] Figure 5 shows a set of chemical structures from some of the examples of the material composition, in particular Figure 5A AGT402 in Figure 5B AGT508 and AGT501 in Figure 5C The AGT502, AGT509, and AGT401, and Figure 5DThe AGT403 and AGT404 in the series. Detailed Implementation

[0065] The following detailed description is provided to assist those skilled in the art of biomedicine. Exemplary embodiments are described in detail. However, these embodiments are merely exemplary. This disclosure is not limited thereto, but is defined by the scope of the appended claims. Modifications and changes can be made to the embodiments described herein without departing from the spirit or scope of this disclosure.

[0066] Industrial applicability

[0067] This invention provides a medically useful composition (pharmaceutical) for treating or slowing the progression of diseases manifested as IgG4-related diseases (IgG4-RD). Diagnosis is based on clinical symptoms and serum antigen-specific IgG4 autoantibodies. IgG4-mediated autoimmune diseases (IgG4-AID) establish IgG4 as a pharmacological target for degradation. These diseases include pemphigus vulgaris, pemphigus foliaceus, LG1 encephalitis, MuSK (IgG4 myasthenia gravis), type 1 autoimmune pancreatitis, interstitial nephritis, Riddle thyroiditis, Mikulicz disease, Thurtner's tumor, inflammatory pseudotumors in multiple sites of the body, mediastinal fibrosis, and some cases of retroperitoneal fibrosis. Khosroshahi & Stone Current Opinion in Rheumatology. 23 (1): 57–66 (January 2011); Stone, Zen, & Deshpande, The New England Journal of Medicine. 366(6), 539–51 (February 2012); Koneczny et al., Frontiers in Immunology. 11, 605214 (January 29, 2021). Other indications for IgG4 include membranous nephropathy, thrombotic thrombocytopenic purpura, chronic inflammatory demyelinating polyneuropathy, and IgG4-related diseases.

[0068] Regardless of the affected body part, the hallmark histopathological features of IgG4-related diseases are: (1) dense infiltration of lymphocytes and IgG4-positive plasma cells; (2) fibrosis arranged in a mat-like pattern; and phlebitis, in which venous channels are blocked by dense infiltration of the venous walls and lumens. See Deshpande et al., Modern Pathology. 25(9), 1181–1192 (May 18, 2012).

[0069]

[0070] pemphigus vulgaris This is an autoimmune vesicular disease affecting the mucous membranes and skin. Positive functional outcomes can be tracked by visually observed improvement in skin condition after IgG4 antibody depletion. A relatively large patient population exists, namely the chronic patient population. Keratinocytes of the skin and mucous membranes are targeted by IgG4 autoantibodies against Dsg1 or Dsg3. These IgG4 autoantibodies have a direct blocking function and lead to loss of cell adhesion. Biomedical data show that a decrease in autoantibody titers is associated with a clinical response.

[0071]

[0072] Muscle-specific kinase (MuSK) positive myasthenia gravis MuSK is a distinct and often more severe form of myasthenia gravis, typically characterized by medullary features and neurological indications. There is a correlation between autoantibody titers and clinical outcomes. IgG4 antibodies (rather than IgG1-3) inhibit the aggregate-dependent binding between MuSK and Lrp4. IgG4 antibodies in MuSK MG patients induce disease without complement. MuSK IgG4 autoantibodies block their interaction with LRP4, thereby impairing a key trophic signaling cascade at the neuromuscular junction and leading to suppressed acetylcholine receptor aggregation, resulting in muscle weakness.

[0073] MuSK myasthenia gravis (MuSK MG) is a therapeutically targeted indication for IgG4 autoantibodies, with the aim of achieving clinical remission in patients. The pathogenesis of MuSK myasthenia gravis involves IgG4 autoantibodies, which are resynthesized at a different rate compared to antigenic IgG4. The slow recovery from the pathogenesis highlights the necessity of continuous and targeted therapeutic interventions.

[0074]

[0075] Anti-LGI-1 encephalitis It is a rare, acute IgG4-related disorder with rapidly resolving indicators, including seizures and cognitive impairment. The disease is treated with rituximab, but this drug has a relatively long onset of action, thus requiring earlier-onset treatment.

[0076] Patients with anti-leucine-rich glioma inactivating protein 1 (LGI1) encephalitis may experience memory impairment, confusion, and faciosacral dystonia-like seizures. LGI1 autoantibodies block the binding of LGI1 to ADAM22, reducing postsynaptic AMPA receptor (AMPAR) levels and leading to neuronal hyperexcitability. Ariño et al., Neurology, 87(8), 759-65 (August 23, 2016) showed that all 57 LGI1 patients with serum available for antibody subclass analysis showed IgG4 antibodies.

[0077]

[0078] Previously recommended treatments for IgG4-related diseases .

[0079] In untreated patients with active disease, the previously recommended first-line agents for inducing remission were glucocorticoids, such as prednisolone 30 to 40 mg / day for 2 to 4 weeks, followed by gradual tapering over three to six months, unless contraindicated. Glucocorticoids are characterized by rapid improvement of clinical features and often cause radiographic regression. When advanced fibrotic lesions lead to irreversible damage, the response to glucocorticoids and other current treatment options may be poor or even nonexistent.

[0080] definition

[0081] For convenience, the meanings of some terms and phrases used in this specification, examples, and appended claims are listed below. As used in this application, each of these terms shall have the meaning described below unless otherwise specified in this specification. Additional definitions are set forth throughout the application. Where a term is not specifically defined in this specification, it is given a meaning generally accepted in the biomedical field for its application in the context of describing the invention. These definitions are helpful in describing embodiments but are not intended to limit the claimed invention.

[0082] The article “a and an” (a / an) refers to the simple meaning of one or more (i.e., at least one) grammatical objects of the article, unless the context otherwise indicates. For example, “an element” means one element or more than one element.

[0083] The term "ABT" has the generally accepted meaning of antibody-binding moiety in the biomedical field. In some embodiments described in this specification, ABT binds to an IgG4 antibody.

[0084] The term "active ingredient" has the meaning provided by the U.S. Food and Drug Administration as any component that provides pharmacological activity or other direct action in the diagnosis, cure, relief, treatment or prevention of a disease, or affects the structure or any function of a human or animal body.

[0085] The term "ADCC" is recognized in the biomedical field as meaning antibody-dependent cell-mediated cytotoxicity. ADCC is a cell-mediated immune defense mechanism in which effector cells of the immune system kill target cells whose membrane surface antigens have been bound by specific antibodies.

[0086] The term “ADCP” is recognized in the biomedical field as antibody-dependent cell-mediated phagocytosis, which is an immune elimination mechanism in which tumor cells are targeted by antibodies to promote the phagocytic clearance of them from the body by immune cells.

[0087] The term "AF488" has the meaning of Alexa Fluor 488, which is recognized in the biomedical field. It is a bright green fluorescent dye that is suitable for excitation by a 488 nm laser line and is used for imaging and flow cytometry.

[0088] The term "AF647" refers to the biomedical term Alexa Fluor 647, a far-infrared dye with an excitation peak at 650 nm and an emission peak at 665 nm. Alexa 647 can be used in flow cytometry, microscopy, and super-resolution microscopy.

[0089] The term "pharmaceutical" has the generally accepted meaning in the biomedical field of a composition of substances used to perform functions. Several useful biomedical functions are described in this specification.

[0090] The term "relief" has a generally accepted meaning in the biomedical field, referring to the process of reducing the severity of a disease's signs or symptoms. Signs or symptoms can be alleviated without elimination. Administration of the compositions or pharmaceutical compositions of the present invention can or is capable of resulting in the elimination of signs or symptoms; however, elimination is not necessary. An effective dose should be expected to reduce the severity of signs or symptoms.

[0091] The terms "effective dose" and "therapeutic effective dose" have the generally accepted meaning in the biomedical field: the amount that effectively achieves its intended purpose. The effect can be detected by any assay method known in the art. The precise effective dose for a subject will depend on the subject's weight, body type, and health condition; the nature and severity of the symptom; and the choice of the therapeutic agent or combination of therapeutic agents used for administration. The therapeutic effective dose for a given situation can be determined through routine experimentation within the skill and judgment of a clinician. In this example, the disease or symptom to be treated is tendinopathy.

[0092] The term "anti-IgG4 antibody" has the meaning of an antibody that selectively binds to IgG4, as recognized in the biomedical field.

[0093] The term "its antigen-binding fragment" has the generally accepted meaning in the biomedical field: (1) a fragment of a complete antibody that binds to the same antigen recognized by a full-length antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, sFv, or other fragments consisting of variable regions; or (2) any synthetic or genetically engineered protein that functions like an antibody by forming a complex with a specific antigen. The term "antigen-binding portion of an antibody" encompasses single-chain antibodies.

[0094] The term "desialylglycoprotein receptor (ASGPR) binding group" has the biomedical meaning of a binding group that binds to the hepatocyte desialyl glycoprotein receptor. The ASGPR binding group selectively binds to the hepatocyte desialyl glycoprotein receptor on the surface of hepatocytes. In several embodiments of this specification, the ASGPR binding group is a component of a bifunctional pharmaceutical agent, acting as a cell receptor binding portion that binds covalently to either a linker group or directly to the IgG4 binding moiety. It is through this ASGPR moiety that bifunctional pharmaceutical agents, such as IgG4 antibodies, complexed with circulating proteins, bind to hepatocytes. After the bifunctional pharmaceutical agent complexed with circulating proteins binds to hepatocytes or other cells, the circulating proteins are taken up into the hepatocytes or other cells via phagocytosis, where they are degraded by lysosomal degradation.

[0095] The term "desialyl glycoprotein receptor (ASGPR)" has the biomedical meaning of lectin, which binds desialylated glycoproteins and glycoproteins that have had sialyl removed to expose galactose residues. These cellular receptors are located on mammalian liver cells and other cells, such as glandular cells of the gallbladder and stomach. ASGPR removes target glycoproteins from circulation.

[0096] When the term "at least one of..." precedes a list of elements, it modifies the entire list of elements, rather than individual elements within the list.

[0097] The term "AT" has the meaning of an antibody moiety recognized in the biomedical field. In some embodiments of this specification, AT binds to IgG4.

[0098] The term "bafloromycin" (bafloromycin A1) refers to a macrolide antibiotic recognized in the biomedical field as an inhibitor of vacuolar H+-ATPase (V-ATPase). It is used in cell biology assays to inhibit autophagy and reduce cell growth in cancer cell lines.

[0099] The term "cell receptor binding moiety" has a generally accepted meaning in the biomedical field. In several embodiments of this specification, the cell receptor binding moiety is a desialoglycoprotein receptor (ASGPR) binding group.

[0100] The term "cell receptor" has a generally accepted meaning in the biomedical field, referring to a protein on the surface of a cell that binds to a compound (e.g., a ligand, such as a protein) in solution or on another cell. Typically, ligand-receptor binding induces one or more biological responses. In this specification, the desialylate glycoprotein receptor (ASGPR) is a cell receptor on the surface of hepatocytes or other cells that binds to desialylate glycoprotein or a derivative thereof.

[0101] The term "Chemistry, Manufacturing and Controls" (Pharmaceutical Quality, CMC) has a generally accepted meaning in the biomedical field, referring to several procedures used to assess the physical and chemical properties of pharmaceutical products, ensure their quality and consistency during manufacturing, and prepare data for submission to regulatory agencies.

[0102] The term "chimeric" has a widely accepted meaning in the biomedical field. Chimeric antibodies are created by fusing a variable domain of one species (such as a mouse) with a constant domain of another species (such as a human). Through this type of biotechnological manipulation, chimeric antibodies retain the antigen specificity and affinity of the exogenous antibody.

[0103] The terms "combination therapy" and "combined therapy" have their generally accepted meaning in the biomedical field, referring to the administration of the composition described herein and at least a second agent as part of a specific treatment regimen intended to provide a beneficial effect through the combined action of these agents. The beneficial effect of the combination may include, but is not limited to, the combined pharmacokinetic or pharmacodynamic effects produced by the combination of the agents. These agents are typically administered in combination over a defined timeframe (usually minutes, hours, days, or weeks, depending on the chosen combination). The term "combination therapy" includes further administration of the aforementioned agents in combination with other bioactive ingredients and non-pharmacological therapies (e.g., surgery or radiation therapy). Where the combination therapy further includes non-pharmacological treatment, the non-pharmacological treatment may be administered at any suitable time, provided that a beneficial effect is achieved from the combined action of the combination of agents. For example, where appropriate, a beneficial effect may still be achieved when the non-pharmacological treatment is temporarily separated from the administration of the agents (potentially by days or even weeks).

[0104] The term "complementarity-determining region (CDR)" has the generally accepted meaning in biomedical fields of the polypeptide region of the antibody heavy or light chain, and is the determining factor in antibody-antigen binding. Each antibody heavy chain has three CDRs. Each antibody light chain has three CDRs, which are usually different from the three CDRs on the antibody heavy chain. A person skilled in the biomedical field uses a standardized numbering method known as the Kabat numbering scheme to count them (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., USA), but other numbering schemes, such as Chothia and IMGT, are also used by a person skilled in the biomedical field.

[0105] The terms “comprises”, “comprising”, “includes”, and “including” specify the presence of the stated features, regions, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0106] The term “desmosomal core glycoprotein 3” (Dsg3) has a generally accepted meaning in the biomedical field as the gene encoding a member of the cadherin protein family. The Dsg3 multiprotein complex is involved in cell adhesion, cytoskeleton organization, and cell signaling. Mice lacking Dsg3 exhibit loss of keratinocyte adhesion, leading to the vesicular phenotype observed in patients with pemphigus vulgaris. Binding to antibodies against Dsg3 results in loss of intracellular adhesion and leads to intraepithelial vesicle formation. The overall homology of Dsg3 between mice and humans is 85.6%. See Buxton et al., The Journal of Cell Biology, 121(3), 481-3 (May 1, 1993); Ishikawa et al., Experimental Dermatology, 9(4), 229-39 (August 2000); and Eming et al., J. Immunol., 193(9), 4391–9 (2022). Monoclonal antibodies targeting Dsg3 bind to epidermal keratinocytes, leading to loss of intercellular adhesion and the formation of intraepithelial vesicles (symptoms of pemphigus vulgaris).

[0107] The term "DMPK" has the recognized meaning of drug metabolism and pharmacokinetics in the biomedical field, which is a discipline that helps identify drugs that may be suitable for further development.

[0108] The term “drug product” has the definition of a finished dosage form as described by the U.S. Food and Drug Administration in 21 CFR § 314.3, such as tablets, capsules, or solutions, which typically, but do not necessarily, contain an active pharmaceutical ingredient associated with one or more other ingredients.

[0109] The term “active drug substance” has the definition of an active ingredient as described by the U.S. Food and Drug Administration in 21 CFR § 314.3, which is intended to provide pharmacological activity or other direct action in the diagnosis, cure, relief, treatment or prevention of a disease, or to affect the structure or any function of the human body, but does not include intermediates used to synthesize such ingredients.

[0110] The term "Fc-III-4c" has a generally accepted meaning in the biomedical field, referring to the polypeptide region within the crystallizable region (Fc region) (tail region) of an antibody fragment.

[0111] The term "Fc-M" has a generally accepted meaning in the biomedical field, referring to the polypeptide region within the crystallizable region (Fc region) (tail region) of an antibody fragment.

[0112] The term "FcB-1" has a generally accepted meaning in the biomedical field, referring to the polypeptide region within the crystallizable region (Fc region) (tail region) of an antibody fragment.

[0113] The term "FcB-2" has a generally accepted meaning in the biomedical field, referring to the polypeptide region within the crystallizable region (Fc region) (tail region) of an antibody fragment.

[0114] The terms "first," "second," "third," etc., have a general meaning in describing various elements, components, regions, layers, and / or segments, and these elements, components, regions, layers, and / or segments should not be limited by these terms. These terms are used only to distinguish one element, component, region, layer, or segment from another element, component, region, layer, or segment. Without departing from the teachings of this embodiment, a first element, component, region, layer, or segment may be referred to as a second element, component, region, layer, or segment.

[0115] The term “Good Laboratory Practice” has the definition described by the U.S. Food and Drug Administration in 21 CFR Part 58 Good Laboratory Practice for Nonclinical Research.

[0116] The term "hepatocyte" is the generally accepted meaning in the biomedical field of the main parenchymal tissue cells of the liver. Hepatocytes account for 55-65% of the liver's mass.

[0117] The term "humanized" has a generally accepted meaning in the biomedical field, referring to proteins, such as antibodies, that are genetically engineered to closely resemble the polypeptide structure of their human homologs. The variable domains of rodent-derived antibodies can be fused with constant domains derived from humans, thus preserving the specificity of the rodent antibody. Human-derived domains do not need to be directly derived from humans; that is, the domain does not need to be synthesized in humans first. Instead, human domains can be generated in rodents whose genomes incorporate human immunoglobulin genes. Antibodies can be partially or fully humanized. In one approach, there are four general steps for humanizing monoclonal antibodies: (1) determining the nucleotide and predicted amino acid sequences of the variable domains of the starting antibody light and heavy chains; (2) designing the humanized antibody, i.e., deciding which antibody framework region to use during the humanization process; (3) the actual humanization method / technique; and (4) transfection and expression of the humanized antibody.

[0118] The term "IC" 50 "It has a generally accepted meaning in the biomedical field, namely the amount, concentration, or dose of a specific test compound that achieves 50% inhibition of the maximum response in a assay measuring such a response."

[0119] The term "IgG antibody" has a universally accepted meaning in the biomedical field. Each IgG molecule consists of a basic four-stranded immunoglobulin structure—two gamma heavy chains and two identical light chains (κ or λ)—and carries two identical antigen-binding sites. There are four subclasses of IgG, each with slightly different H chains but distinct biological properties.

[0120] The term "IgG1 antibody" has the meaning of IgG antibody as recognized in the biomedical field, where the C region of the Igγ-1 chain is a protein encoded by the IGHG1 gene in humans.

[0121] The term "IgG2 antibody" has the meaning of IgG antibody as recognized in the biomedical field, where the C region of the Igγ-2 chain is a protein encoded by the IGHG2 gene in humans.

[0122] The term "IgG4 binding moiety" has a generally accepted meaning in the biomedical field, referring to the portion of a binding protein (such as an IgG antibody or a fragment thereof) that binds to an IgG4 antibody.

[0123] The term "IgG4 antibody" refers to the biomedical term for IgG antibodies, where the C region of the Igγ-4 chain is a protein encoded by the IGHG4 gene in humans. IgG4 has virtually no effector function. IgG4 does not fix complement.

[0124] The term "IVIG" has the meaning of intravenous immunoglobulin (IVIG) as recognized in the biomedical field.

[0125] Term "K" D "It has a recognized meaning in the biomedical field, namely, the measured equilibrium dissociation constant between a compound or ligand and a protein or the binding domain of a protein."

[0126] The term "LGI1" has the meaning of a neuronal antigen recognized in the biomedical field as involved in synaptic function, because it is part of a voltage-gated potassium channel complex and is involved in connecting transmembrane proteins across the synaptic cleft.

[0127] The term "connector portion" has the generally accepted meaning in the biomedical field of a portion of a chemical compound that connects one part of the chemical compound to another part of the same compound. In several embodiments of the present invention, the connector portion connects an anti-IgG4 IgG antibody portion to a cell receptor binding portion.

[0128] The term “MoDE” has the proprietary meaning of molecular degrading agent. See International Patent Publication No. WO 2019 / 199634 (Yale University) and International Patent Publication No. WO 2019 / 199621 (Yale University).

[0129] The term "part" has a biomedical meaning, referring to a defined chemical group or entity having a specific structure or activity. A part typically refers to a portion of a molecule; for example, in an ester RCOOR', the alcohol part is RO−. In some embodiments, a part of a pharmaceutical agent (e.g., a target agent, peptide agent, antibody agent, etc.) retains one or more or all of the desired structural features, properties, functions, and / or activities of the compound. In some embodiments, a cell receptor-binding part may bind to a target, optionally in a manner equivalent to its corresponding target binder; in some embodiments, a target agent part maintains one or more desired structural features, properties, functions, and / or properties equivalent to its corresponding target agent; in some embodiments, an antibody agent part maintains one or more desired structural features, properties, functions, and / or properties equivalent to its corresponding antibody agent, such as 3D structure, antigen specificity, antigen binding capacity, and / or immune function, etc. In some embodiments, portions of the agent (e.g., target portion, peptide portion, antibody portion, etc.) are monovalent (for monovalent portions), divalent (for divalent portions), or polyvalent (for polyvalent portions) groups of the agent, such as target (for target portion), peptide (for peptide portion), antibody (for antibody portion), etc. In some embodiments, the monovalent group is formed by removing a monovalent portion (e.g., hydrogen, halogen, another monovalent group, such as alkyl, aryl, etc.) from the compound / agent. In some embodiments, the divalent or polyvalent group is formed by removing one or more monovalent (e.g., hydrogen, halogen, monovalent group such as alkyl, aryl, etc.), divalent, and / or polyvalent portions from the compound / agent. In some embodiments, the group is formed by removing a hydrogen atom. In some embodiments, the portion is monovalent. In some embodiments, the portion is divalent. In some embodiments, the portion is polyvalent.

[0130] The term "monotherapy" has the generally accepted meaning in the biomedical field of administering a single active or therapeutic compound to a subject. Monotherapy typically involves administering a therapeutically effective amount of an active composition.

[0131] The term "multimodal antibody therapy enhancer" (MATE or MATES) has a proprietary meaning. See International Patent Publication No. WO 2021 / 102052 (Kleo Pharmaceuticals).

[0132] The term "nanobody" has a generally accepted meaning in the biomedical field.

[0133] The term "antibody" refers to immunoglobulin molecules and the immunoactive portion of immunoglobulin molecules, i.e., molecules containing antigen-binding sites that specifically bind to antigens. The term also refers to antibodies composed of two immunoglobulin heavy chains and two immunoglobulin light chains, as well as various forms including full-length antibodies and their antigen-binding portions; including, for example, immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-transplanted antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single-domain antibodies, biantibodies, nanobodies, multispecific (e.g., trispecific) antibodies, bispecific antibodies, anti-idiotype antibodies, their functional active epitope binding portions, and / or bifunctional hybrid antibodies.

[0134] The term "on" has a general meaning. When an element is said to be on another element, it can be in direct contact with the other element or there can be an intermediate element between the two elements. When an element is said to be "directly on another element," there is no intermediate element.

[0135] As used in this specification, the term "or" means "and / or". As used in this specification, the term "and / or" includes any and all combinations of one or more of the relevant listed items.

[0136] The term "other degrading cells" has a generally accepted meaning in the biomedical field. Desialyl glycoprotein receptors (ASGPRs) are found on glandular cells in the gallbladder and stomach.

[0137] The term "partially humanized" has a generally accepted meaning in the biomedical field: proteins, such as antibodies, are genetically engineered to make their polypeptide structures more similar to human homologs. The variable domains of rodent-derived antibodies can be fused with constant domains from human sources, thus preserving the specificity of the rodent antibody. The human-derived domains do not need to originate directly from humans; that is, they do not necessarily have to be synthesized first in humans. Instead, human domains can be generated in rodents whose genomes incorporate human immunoglobulin genes. Antibodies can be partially or fully humanized.

[0138] The term "pharmaceuticalally acceptable excipient" has the meaning of an excipient recognized in the biomedical field as suitable for the preparation of pharmaceutical compositions that are generally safe, non-toxic, and not biologically or otherwise undesirable, and includes excipients acceptable for veterinary or human pharmaceutical use. As used in the specification and claims, "pharmaceuticalally acceptable excipient" includes one or more such excipients. A detailed discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences, 23rd edition (Elsevier, 2020).

[0139] The term "pharmaceutically acceptable" has the meaning in the biomedical field of compounds, anions, cations, materials, compositions, carriers, and / or dosage forms that, within reasonable medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0140] The term "protein-binding portion" has the meaning, as is generally accepted in the biomedical field, of a region of a chemical composition that specifically binds to a protein (e.g., a specific protein) (e.g., a polypeptide region of the chemical composition).

[0141] The term "rIgG" has the meaning of recombinant human IgG as recognized in the biomedical field.

[0142] The term "ROC" has the meaning of receiver operating characteristic curve, which is generally accepted in the biomedical field.

[0143] The terms "subject" and "patient" have their generally accepted meanings in the biomedical field. The term "patient" includes human and other mammalian subjects who receive preventative or therapeutic treatment.

[0144] The term "TBT" in this specification has the meanings of target-binding portion and cell receptor-binding portion. In some embodiments of this specification, TBT binds to ASGPR.

[0145] The terms “treating” and “treatment” have the generally accepted meaning in the biomedical field of managing and caring for a patient in response to a disease, symptom, or condition, and include the administration of the compositions described in this specification to alleviate or eliminate the symptoms or complications of a disease, symptom, or condition.

[0146] The term "universal antibody-binding moiety" has a generally accepted meaning in the biomedical field, referring to the polypeptide region of an antibody-binding protein that binds to a class of antibodies rather than a specific set of antibodies.

[0147] The term "VHH" has a generally accepted meaning in the biomedical field: the variable heavy chain domain of a heavy chain antibody derived from camelid antibodies. VHH has nine β-sheets, forming a cylindrical structure. Several regions may participate in binding. Human IgG is approximately 150 kilodaltons. Cameloid IgG is approximately 90 kilodaltons. The VHH single-chain domain is approximately 15 kilodaltons. The most important regions are 4 > 1 and 2 (with the greatest difference in absorption). The least important regions are 3 and 5 (with very small differences in absorption).

[0148] The following describes some embodiments with reference to structures and schemes to explain the aspects described.

[0149] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the biomedical field.

[0150] This specification does not cover methods for cloning humans, methods for altering the germline genetic identity of humans, the use of human embryos for industrial or commercial purposes, or processes for altering the genetic identity of animals that may cause suffering to the animals and for which such processes do not provide substantial medical benefit to humans or animals.

[0151] Methods for administering bifunctional degradative agents to subjects and measuring the results .

[0152] How to identify subjects to be administered bifunctional degradative agents Serum immunoglobulin G4 (IgG4) levels are frequently elevated, but this is not always the case. However, a blood laboratory level of IgG4 greater than 135 is considered an evolving diagnostic criterion for suspected disease.

[0153] IgG4 levels vary among healthy individuals and even within individual patients. In a retrospective cohort study, baseline concentrations of serum IgG4, IgE, and blood eosinophils independently predicted the risk of relapse after treatment. Higher baseline values ​​were associated with a greater risk of relapse and a shorter time to relapse. (Wallace et al., Rheumatology, 55(6), 1000–1008 (June 6, 2016)).

[0154] A definitive diagnosis is important because treatment approaches differ for IgG4 diseases and those that mimic them. Satou et al., Pathol.Int., 70(7), 391–402 (July 2020). While serum IgG4 levels may be indicative, a definitive histological diagnosis is required to confirm IgG4 disease and rule out similar symptoms. Chen et al., Haematologica, 104(3), 444 (March 2019). The sensitivity of IgG4 levels in diagnosing IgG4-related diseases varies depending on the assay used, the number of organs affected, and possibly the patient's geographic origin. See Acharya et al., J. Community Hosp. Intern. Med. Perspect., 14(3), 113–118 (May 7, 2024).

[0155] How to apply The optimal mode of administration depends on where the treatment takes place, whether it is in a hospital or an outpatient clinic.

[0156] Therapeutic agents can be delivered via subcutaneous injection, for example, by administering a 6 mL subcutaneous infusion or by further optimizing the dose concentration and volume for subcutaneous administration. Alternative formulations, such as VHH-Fc fusion proteins, can reduce the amount that needs to be administered. In one embodiment, the non-Fc fusion VHH molecule exhibits a lower molecular weight, which is advantageous for subcutaneous formulations, with an estimated target dose of approximately 125 mg.

[0157] Administering in divided doses, such as four 125 mg doses, can be a method for administering the full 500 mg dose. Alternative formulations may require a loading dose to achieve sufficient IgG4 depletion.

[0158] The inventors conducted a series of assays in mice and rats to gain insights into subcutaneous and intravenous administration routes using repeated dosing regimens to demonstrate the bioavailability of the administered drug in subjects.

[0159] A method for measuring the removal of IgG4 antibodies from a patient or subject. Antibody depletion leads to functional outcomes. The inventors have performed in vivo consumption assays to measure IgG4 depletion. See the examples provided in this specification. Other assays for measuring IgG4 in blood circulation and functional outcomes of IgG4-related diseases are known to those skilled in the art of biomedicine.

[0160] Chemical structure of a substance composition (pharmaceutical) .

[0161] In one embodiment, the present invention provides a substance composition (pharmaceutical) comprising:

[0162] IgG4 binding site,

[0163] The cell receptor binding portion binds to hepatocytes or other degrading cells via the desialylate glycoprotein receptor (ASGPR) on the surface of hepatocytes of the patient or subject, or via other cell receptors on the surface of degrading cells.

[0164] The connector portion connects the antibody portion and the cell receptor binding portion.

[0165] In some embodiments, the present invention provides a substance composition (pharmaceutical) having the following structure:

[0166]

[0167] [CPBM] is the IgG4 antibody binding portion;

[0168] [CRBM] is the cell receptor binding moiety;

[0169] Each [CON] is an optional connector chemical part, which, when present, connects directly to [CPBM] or [CRBM] or connects [LINKER] to [CPBM] or [CRBM], and

[0170] [LINKER] is a chemical moiety covalently attached to one or more [CRBM] or [CPBM] groups, or

[0171] Its pharmaceutically acceptable salts, stereoisomers, solvates, or polymorphs.

[0172] In some embodiments, the present invention provides a composition (pharmaceutical) having a structure selected from the Markush structural group consisting of:

[0173] R CN −(Xaa)y−R CC ,

[0174]

[0175] [AGN101],

[0176]

[0177] [AGN102],

[0178]

[0179] [AGN103], or

[0180]

[0181] [AGN104] or its salts. In these structures, a, b, and c can independently be integers of 1 or greater. In some embodiments, each cell receptor-binding portion independently has −(R CN −(Xaa)y−R CC The structure of ) or its salt form.

[0182] In some embodiments, the agent has the structure of formula AGN101:

[0183]

[0184] [AGN101] or its salt, wherein:

[0185] Each of a, b, and c is independently between 1 and 200;

[0186] Each AT is independently an IgG4 binding moiety;

[0187] L is the connector part; and

[0188] Each TBT is an independent cell receptor-binding moiety.

[0189] The IgG4 binding portion is (1) an anti-IgG4 antibody or its antigen-binding fragment, or (2) an anti-IgG4 VHH affinity ligand variant or its antigen-binding fragment.

[0190] In some embodiments, the agent has the structure of formula AGN102:

[0191]

[0192] [AGN102] or its salt, wherein:

[0193] Each of a and b is independently 1-200;

[0194] Each AT is independently an IgG4 binding moiety;

[0195] L is the connector part; and

[0196] Each TBT is an independent cell receptor-binding moiety.

[0197] The IgG4 binding portion is (1) an anti-IgG4 antibody or its antigen-binding fragment, or (2) an anti-IgG4 VHH affinity ligand variant or its antigen-binding fragment.

[0198] In some embodiments, the agent comprises one and no more than one IgG4 binding moiety. In some embodiments, one or more IgG4 binding moiety binds to a linker moiety. In some embodiments, a is 1. In some embodiments, a is 2 or greater. In some embodiments, one and no more than one cell receptor binding moiety is bonded to a linker moiety. In some embodiments, b is 1. In some embodiments, two or more cell receptor binding moiety are bonded to a single linker moiety. In some embodiments, b is 2 or greater. In some embodiments, the agent comprises one and no more than one cell receptor binding moiety. In some embodiments, c is 1. In some embodiments, b is 1 and c is 1. In some embodiments, a is 1, b is 1, and c is 1. In some embodiments, the agent comprises two or more IgG4 binding moiety. In some embodiments, b is 2 or greater, and c is 1. In some embodiments, b is 2 or greater, and c is 2 or greater. In some embodiments, b is 1, and c is 2 or greater.

[0199] In some embodiments, b is 1. In some embodiments, c is 1. In some embodiments, c is two or more. In some embodiments, c is 2. Those skilled in the biomedical field know several techniques for conjugating antibody portions to target-binding portions, such as certain techniques for preparing antibody-drug conjugates according to this specification. In some embodiments, the target-binding portion is linked to the antibody portion via certain types of groups and / or amino acid residues. In some embodiments, the target-binding portion is optionally linked to a lysine residue via a linker portion. In some embodiments, the target-binding portion is optionally linked to a cysteine ​​residue via a linker portion. In some embodiments, the target-binding portion is optionally linked to a non-natural amino acid residue via a linker portion. In some embodiments, the present invention provides techniques for selectively linking a target-binding portion to certain specific amino acid residues optionally via a linker portion. In some embodiments, the provided techniques selectively link the target-binding portion to certain types of amino acid residues (e.g., lysine residues) via a linker portion. In some embodiments, the provided techniques selectively link the target-binding portion to a specific site of the antibody portion via a linker portion. In some embodiments, the provided techniques selectively link the target-binding portion to certain types of amino acid residues at the site via a linker portion. In some embodiments, the target-binding portion is optionally linked via a linker portion to K246 and K248 of the IgG1 heavy chain and their corresponding amino acid residues. In some embodiments, the target-binding portion is optionally linked via a linker portion to K251 and K253 of the IgG2 heavy chain and their corresponding amino acid residues. In some embodiments, the target-binding portion is optionally linked via a linker portion to K239 and K241 of the IgG4 heavy chain and their corresponding amino acid residues. In some embodiments, the cell receptor-binding portion is optionally linked via a linker to a specific amino acid residue or site. In some embodiments, each cell receptor-binding portion is optionally linked independently via a linker to a specific amino acid residue or site. Those skilled in the biomedical field will know that antibody agents can contain more than one site (e.g., one site on each chain in more than one chain (e.g., one or each heavy chain). In some embodiments, the antibody portion comprises two heavy chains, and one or both of the amino acid residues or their corresponding amino acid residues are optionally linked independently via a linker to the cell receptor-binding portion. In some embodiments, one and no more than one are linked. In some embodiments, c is 1. In some embodiments, both are linked. In some embodiments, c is 2. In some embodiments, the two target mating portions or the two connector portions are identical.

[0200] In some embodiments, each cell receptor-binding portion of the drug is identical. In some embodiments, each connector portion connecting the cell receptor-binding portion to the antibody portion is identical.

[0201] In some embodiments, the material composition may be selected from the following compounds:

[0202]

[0203] IgG4 binding site

[0204] In some embodiments, the IgG4 binding portion is linked to the antibody portion via certain types of groups or amino acid residues. In some embodiments, the IgG4 binding portion is optionally linked to a lysine residue via a linker portion. In some embodiments, the IgG4 binding portion is optionally linked to a cysteine ​​residue via a linker portion. In some embodiments, the IgG4 binding portion is optionally linked to a non-natural amino acid residue via a linker portion. In some embodiments, the present invention provides techniques for selectively linking the IgG4 binding portion to certain amino acid residues via linker portions. In some embodiments, the provided techniques selectively link the IgG4 binding portion to certain types of amino acid residues (e.g., lysine residues) via linker portions. In some embodiments, the provided techniques selectively link the IgG4 binding portion to a site on the antibody portion via linker portions. In some embodiments, the provided techniques selectively link the IgG4 binding portion to certain types of amino acid residues at the site via linker portions. In some embodiments, the IgG4 binding portion is optionally linked to K246 and K248 of the IgG1 heavy chain and their corresponding amino acid residues via linker portions. In some embodiments, the IgG4 binding portion is optionally linked to K251 and K253 of the IgG2 heavy chain and their corresponding amino acid residues via linker portions. In some embodiments, the IgG4 binding portion is optionally linked via a linker portion to K239 and K241 of the heavy chain and their corresponding amino acid residues. In some embodiments, the cell receptor binding portion is optionally linked via a linker to a specific amino acid residue or site. In some embodiments, each cell receptor binding portion is optionally independently linked via a linker to a specific amino acid residue or site.

[0205] Those skilled in the art of biomedicine will know that antibody agents can contain more than one site (e.g., one site on each of more than one chain (e.g., one or each heavy chain)). In some embodiments, the antibody portion comprises two heavy chains, and one or both of the amino acid residues or their corresponding amino acid residues are optionally independently linked to the cell receptor binding portion via a linker. In some embodiments, one and no more than one linker are used. In some embodiments, c is 1. In some embodiments, both are linked. In some embodiments, c is 2. In some embodiments, the two IgG4 binding portions or the two linker portions are identical.

[0206] In another embodiment, the IgG4 binding portion is an IgG4-binding antibody or its antigen-binding fragment.

[0207] In another embodiment, the IgG4 binding portion is a variant of ABT301 (SEQ ID NO: 1).

[0208] In another embodiment, the IgG4 binding portion is a complete IgG antibody, wherein the heavy chain contains the three CDR regions of the VHH affinity ligand. The full-length human IgG can be natural and have variable glycosylation.

[0209] In some embodiments, the IgG4 binding portion comprises a portion selected from the Markush group, which consists of one or more amino acid residues, a peptide portion, a cyclic peptide portion, a peptide containing one or more natural amino acid residues, and a peptide containing one or more non-natural amino acid residues.

[0210] The IgG4 binding moiety may be or may contain ABT101 or a salt thereof. In some embodiments, ABT101 is an IgG4 binding moiety. Each IgG4 binding moiety in the pharmaceutical preparation may be the same IgG4 binding moiety or a salt thereof. The IgG4 binding moiety may be an anti-IgG4 VHH nanobody or an antigen-binding variant thereof.

[0211] In some embodiments, ABT101 is a universal antibody-binding moiety. In some embodiments, ABT101 is a universal antibody-binding moiety that can bind to IgG4 antibodies with different Fab regions. In some embodiments, ABT101 is a universal antibody-binding moiety that binds to Fc regions (e.g., Fc regions that bind to Fc receptors). VHH nanobodies are a universal binder because IgG4 binding of VHH nanobodies occurs via IgG4 Fc.

[0212] In one embodiment, the IgG4 binding moiety is human IgG4 Fc protein, untagged, ACRO Biosystems, catalog number IG4-H5205, which can be produced by HEK293 expression and has amino acids Glu 99-Lys 327.

[0213] In some embodiments, the IgG4 binding portion comprises a portion selected from the Markush group, which consists of one or more amino acid residues, a peptide portion, a cyclic peptide portion, a peptide containing one or more natural amino acid residues, and a peptide containing one or more non-natural amino acid residues.

[0214] The IgG4 binding moiety may be or may include:

[0215]

[0216] [ABT101]

[0217] Or its salt form.

[0218] In some embodiments, ABT101 is an IgG4 binding moiety. Each IgG4 binding moiety in the pharmaceutical preparation may be the same IgG4 binding moiety or a salt thereof.

[0219] Several IgG4 binding moieties may be used in accordance with the teachings of this specification. Some IgG4 binding moieties and techniques for identifying or evaluating IgG4 binding moieties are described in International Patent Publications WO 2019 / 023501 and WO2019 / 136442, the entire contents of each of which are incorporated herein by reference. Those skilled in the biomedical field will recognize that other techniques in the biomedical field may be suitable for identifying or evaluating IgG4 binding moieties according to this specification. In some embodiments, the IgG4 binding moieties comprise one or more amino acid residues, each amino acid residue being independently natural or non-natural.

[0220] In some embodiments, the IgG4 binding portion is or contains R c −(Xaa)z− or its salt form, wherein each variable is as described in this specification. In some embodiments, the IgG4 binding portion is or contains ABT101 or its salt form, wherein each variable is as described in this specification.

[0221] In some embodiments, the protein-binding portion is or includes R c −(Xaa)z− or its salt form, wherein each variable is as described in this specification. In some embodiments, the protein-binding portion is or contains ABT101 or its salt form, wherein each variable is as described in this specification.

[0222] In some embodiments, the IgG4 binding portion is or contains R c −(Xaa)z− or its salt form, wherein each variable is as described in this specification. In some embodiments, the IgG4 binding portion is or contains ABT101 or its salt form, wherein each variable is as described in this specification. In some embodiments, the IgG4 binding portion is R c -(Xaa)z− or ABT101 or its salt form, and is or contains a peptide unit.

[0223] In some embodiments, amino acid residues may form bridges, for example, links formed by side chains, optionally through a linker portion (e.g., L); for example, as in many polypeptides, cysteine ​​residues may form disulfide bridges.

[0224] In some embodiments, the peptide unit comprises amino acid residues, such as positively charged amino acid residues, Xaa, at a physiological pH of about 7.4. P In some embodiments, the peptide unit comprises R. In some embodiments, at least one Xaa is R.

[0225] Those skilled in the biomedical field know that amino acid residues can be replaced by other amino acid residues with similar properties, for example, a Xaa. H For example, Val, Leu, etc., can be used by another Xaa H Replacements, such as Leu, Ile, Ala, etc., a Xaa A It can be another Xaa A Replace, a Xaa P It can be another Xaa P Replace, a Xaa N It can be another Xaa N Replace, a Xaa L It can be another Xaa L Replacement, etc.

[0226] In some embodiments, the IgG4 binding portion is or includes an optionally substituted portion, as shown in the table provided on pages 48 to 60 of International Patent Publication WO2019 / 023501, which, along with the patent publication, is incorporated herein by reference.

[0227] In some embodiments, useful techniques for developing or evaluating antibody-binding moieties are described in the following: for example, Alves, Langmuir, 28, 9640−9648 (2012), Choe et al., Materials, 9, 994 (2016), Gupta et al., Nature Biomedical Engineering, 3, 917–929 (2019), Muguruma et al., ACSOmega, 4, 14390−14397 (2019), Yamada et al., Angew. Chem. Int., Ed. Engl., 58(17), 5592–5597 (April 16, 2019), Kruljec et al., Bioconjugate Chem., 28(8): 2009–2030 (2017), such as Fabsorbent, triazines, etc.; Kruljec et al., Bioconjugate Chem., References 29(8),2763-2775 (2018), International Patent Publication WO 2012 / 017021, etc., are incorporated herein by reference in their entirety.

[0228] In some embodiments, the IgG4 binding portion is an affinity substance described in International Patent Publication WO 2018 / 199337, the respective affinity substance of which is incorporated herein by reference.

[0229] In some embodiments, the IgG4 binding portion is or comprises, for example, an adaptor protein agent as described in Hui et al., Bioconjugate Chem., 26, 1456−1460 (2015). In some embodiments, when used according to this specification, the adaptor protein does not require reactive residues (e.g., BPA) to achieve one or more of the advantages.

[0230] In some embodiments, the antibody-binding portion may be selected from the following compounds:

[0231]

[0232] Characterizing the antibody-binding portion .

[0233] Many techniques can be used to identify, evaluate, or characterize antibody-binding moieties, such as those described in International Patent Publication WO 2019 / 023501, the techniques of which are incorporated herein by reference. In some embodiments, the antibody-binding moieties are those that selectively bind to IgG and, when used in the provided techniques, provide and / or stimulate ADCC and / or ADCP, such as small molecule moieties, peptide moieties, nucleic acid moieties, etc. In some embodiments, peptide display techniques (e.g., phase display, non-cellular display, etc.) can identify antibody-binding moieties. In some embodiments, the antibody-binding moieties are those that can bind to IgG and optionally compete with known antibody conjugates such as protein A, protein G, protein L, etc. (e.g., small molecule moieties, peptide moieties, nucleic acid moieties, etc.).

[0234] Those skilled in the biomedical field will know that antibodies possessing various properties and activities, such as antibodies recognizing different antigens or having optional modifications, can be targeted by the antibody-binding moiety described herein. In some embodiments, such antibodies include antibodies administered to a subject, for example, for therapeutic purposes. In some embodiments, the antibody-binding moiety described herein can bind antibodies against different antigens and can be used to conjugate the moiety of interest to several antibodies.

[0235] In some embodiments, the IgG4 binding portion is or includes a mediatope pharmaceutical portion. In some embodiments, the mediatope pharmaceutical portion is described, for example, in U.S. Patent Publication 2019 / 0111149.

[0236] In some embodiments, the IgG4 binding portion may bind to human IgG. In some embodiments, the IgG4 binding portion may bind to an antibody selected from the Markush antibody group, which consists of rabbit IgG, IgG1, IgG2, IgG3, and IgG4. In some embodiments, the IgG4 binding portion binds to IgG1, IgG2, and IgG4.

[0237] In some embodiments, the reference technology is used As a non-antibody binding portion. In some embodiments, the chemical groups used as the non-antibody binding portion in the reference art are selected from the Markush group of chemical groups, which consists of CH3−, CH3C(O)−, CH3C(O)NH−, CH3C(O)NHCH2−, CH3CH2−, CH3CH2NH−, and CH3CH2NHC(O)−.

[0238] In some embodiments, the antibody-binding portion (e.g., the antibody-binding site) is at approximately 1 mM -1 pM or smaller K DBinding to a target (e.g., an antibody agent with an antibody-binding moiety). In some embodiments, K D It is approximately 1 mM, 0.5 mM, 0.2 mM, 0.1 mM, 0.05 mM, 0.02 mM, 0.01 mM, 0.005 mM, 0.002 mM, 0.001 mM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM or smaller. In some embodiments, K D The affinity is selected from the Markush affinity group consisting of the following: about 1 mM or less, about 0.5 mM or less, about 0.1 mM or less, about 0.05 mM or less, about 0.01 mM or less, about 0.005 mM or less, about 0.001 mM or less, about 500 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less, and about 1 nM or less. In some embodiments, the antibody binding portion is K as described in this specification. D Binding to IgG antibody agents.

[0239] Those skilled in the biomedical field will know that antibodies with various properties and activities, such as antibodies recognizing different antigens, having optional modifications, etc., can be recruited by the antibody-binding portion described herein. In some embodiments, such antibodies include antibodies administered to a subject, for example, for therapeutic purposes. In some embodiments, antibodies recruited via the antibody-binding portion include antibodies against different antigens. In some embodiments, antibodies recruited via the antibody-binding portion include antibodies against antigens not present on the surface or cell membrane of target cells. In some embodiments, antibodies recruited by the antibody-binding portion comprise antibodies that do not target antigens present on the surface or cell membrane of a target. In some embodiments, antigens on the surface of target cells may interfere with the structure, conformation, and / or one or more properties and / or activities of recruited antibodies that bind to such antigens. In some embodiments, the recruited antibody is an antibody in IVIG. In some embodiments, IVIG may be administered before, simultaneously with, or after a pharmaceutical agent or composition. Antibodies of several types of immunoglobulin structures can be recruited. In some embodiments, one or more subclasses of IgG are recruited. In some embodiments, the recruited antibody is selected from the Markush antibody class group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the recruited antibody is or contains IgG1 and IgG2. In some embodiments, the recruited antibody is or comprises IgG1, IgG2, and IgG4. In some embodiments, the recruited antibody is or comprises IgG1, IgG2, IgG3, and IgG4. The recruited antibody can interact with several types of receptors, such as receptors expressed by several types of immune cells. In some embodiments, the recruited antibody can effectively interact with several types of Fc receptors and provide the desired immune activity. In some embodiments, the recruited antibody can recruit immune cells. In some embodiments, the recruited antibody can effectively interact with hFcγRIIIA. In some embodiments, the recruited antibody can effectively interact with hFcγRIIIA on macrophages. In some embodiments, macrophages are recruited to provide ADCC and / or ADCP activity against viruses (e.g., SARS-CoV-2 virus) and / or cells infected therewith. In some embodiments, NK cells are recruited to provide immune activity. In some embodiments, the recruited antibody can effectively interact with hFcγRIIA. In some embodiments, the recruited antibody can effectively interact with hFcγRIIA on dendritic cells. In some embodiments, the antibody portion of the pharmaceutical preparation described herein includes one or more properties, structures, and / or activities of the recruited antibody described herein.

[0240] Fc mutation strategies to enhance FcRn binding .

[0241] There are many publicly available methods and mutants to increase the binding of Fc to FcRn. See Table 11. An older abstract was submitted in Strohl, Optimization of Fc-mediated effector functions of monoclonal antibodies. Current Opinion in Biotechnology, 20(6), 685-691 (2009).

[0242]

[0243] For an example of introducing the YTE region into the protein (M252Y / S254T / T256E), see Acqua et al., Increasing the affinity of a human IgG1 for the neonatal Fc receptor: Biological consequences. The Journal of Immunology 169(9), 5171-5180 (2002). For a recent example of binding LALA and YTE, see Cobb et al., A combination of two human neutralizing antibodies prevents SARS-CoV-2 infection in rhesus macaques. bioRxiv, 2021-09 (2021). Other references with engineering details are also available.

[0244] For an example of introducing the LS region into a protein (M428L / N434S), see Zalevsky et al. Nature Biotechnology, 28(2), 157-159 (2010).

[0245] The linker portion and its amino acids

[0246] In some embodiments, the connector portion has a structure of type LNK101:

[0247] NH(R a1 )−L a1 −C(R a2 (R) a3 )−L a2 -COOH

[0248] [LNK101] or a salt thereof, wherein:

[0249] R a1R a2 R a3 Each of them is independently −L a -R';

[0250] L a1 and L a2 Each of them is L independently. a ;

[0251] Each L a Independently covalent bonds, or selected from C1-C bonds having 1-5 heteroatoms. 20 Aliphatic or C1-C 20 A heteroaliphatic optionally substituted divalent group, wherein one or more methylene units of said group are optionally and independently substituted with: −C(R')2−, −Cy−, −O−, −S−, −S−S−, −N(R')−, −C(O)−, −C(S)−, −C(NR')−, −C(O)N(R')−, −N(R')C(O)N(R')−, −N(R')C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R')−, −C(O)S− or −C(O)O−;

[0252] Each −Cy− is independently a optionally substituted divalent monocyclic, bicyclic, or polycyclic group, wherein each monocyclic group is independently selected from C… 3-20 Alicyclic rings, C 6-20 Aryl ring, 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-20 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;

[0253] Each R' is independently −R, −C(O)R, −CO2R, or −SO2R;

[0254] Each R is independently an −H or optionally substituted group, wherein the optionally substituted group is selected from C. 1-30 Aliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon 1-30 Aliphatic, C 6-30 Aryl, C 6-30 Aryl aliphatic, having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. 6-30 Aryl heteroaliphatic, 5-30 membered heteroaryl groups having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclic groups having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or

[0255] The two R groups optionally and independently form a covalent bond together, or:

[0256] Two or more R groups on the same atom optionally and independently form, together with the atom, a 3-30 member monocyclic, bicyclic, or polycyclic ring having 0-10 independently substituted heteroatoms selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, in addition to the atom; or

[0257] Two or more R groups on two or more atoms optionally and independently form, together with the atoms between them, a 3-30 member monocyclic, bicyclic or polycyclic ring having 0-10 independently substituted heteroatoms selected from oxygen, nitrogen, sulfur, phosphorus and silicon, excluding the intervening atom.

[0258] In some embodiments, the amino acid residues have −N(R a1 )−L a1 −C(R a2 (R) a3 )−L a2 The structure of −COO− or its salt form.

[0259] In some embodiments, an amino acid analog is a compound in which the amino and / or carboxylic acid groups are independently substituted with alternatively substituted aliphatic or heteroaliphatic portions. As known to those skilled in the biomedical art, many amino acid analogs that mimic the structure, properties, and / or function of amino acids are described in the biomedical field and can be used according to this specification, for example, in several sections. In some embodiments, one or more peptide groups are optionally and independently substituted with non-peptide groups. In some embodiments, the amino acid portion of a polypeptide or peptide is partially replaced by an amino acid analog.

[0260] In some embodiments, portions are optionally connected to each other via a connector portion. In some embodiments, reactive groups, such as RG, are connected via a connector, such as L... RM A cell receptor-binding portion, such as a TBT connector. In some embodiments, the portion, such as LG, may also include one or more connectors, such as L. LG1 L LG2 L LG3 L LG4 etc., to connect several parts. In some embodiments, L LG This is the connector portion described in this specification. In some embodiments, L LG1 This is the connector portion described in this specification. In some embodiments, L LG2 This is the connector portion described in this specification. In some embodiments, L LG3 This is the connector portion described in this specification. In some embodiments, L LG4 This is the connector portion described in this specification. In some embodiments, L RMThis is the connector portion described in this specification. In some embodiments, L PM It is L. In some embodiments, L PM This is the connector portion described in this specification. In some embodiments, L PM It is L.

[0261] Several types of connector portions may be used in accordance with this specification and / or for several purposes, such as those for antibody-drug conjugates, etc.

[0262] The connector portion can be bivalent or polyvalent, depending on its intended use. In some embodiments, the connector portion is bivalent. In some embodiments, the connector is polyvalent and connects more than two portions.

[0263] In some embodiments, the connector portion, for example L z (where z represents superscript text; for example, L) PM L RM L LG L LG1 "etc." means "is or contains L".

[0264] In some embodiments, L is a covalent bond or a divalent or polyvalent optionally substituted straight or branched chain C. 1-100 A group comprising one or more aliphatic, aryl, heteroaliphatic, heteroaromatic, or any combination thereof having 1-20 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced by: C 1-6 Alkylene, C 1-6 alkenyl group, divalent C with 1-5 heteroatoms 1-6 heteroaliphatic groups, , −Cy−, −C(R')2−, −O−, −S−, −S−S−, −N(R')−, −C(O)−, −C(S)−, −C(NR')−, −C(O)N(R')−, −C(O)C(R ')2N(R')−, −N(R')C(O)N(R')−, −N(R')C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R')−, −C(O)S−, −C(O )O−、−P(O)(OR')−、−P(O)(SR')−、−P(O)(R')−、−P(O)(NR')−、−P(S)(OR')−、−P(S)(SR')−、−P(S)(R')−、−P(S)(NR')−、−P(R')−、−P(OR')−、−P(SR')−、−P(NR')−、amino acid residues or −[(−O−C(R')2−C(R')2−) n]−, where n is 1-20. The linker optionally has a cyclic group Cy as defined below and a reactive group RG as defined below. In some embodiments, each amino acid residue is independently an amino acid having the structure of formula LNK101 or a salt thereof. In some embodiments, each amino acid residue independently has −N(R a1 )−L a1 −C(R a2 (R) a3 )−L a2 The structure of -CO− or its salt form.

[0265] In some embodiments, L is divalent. In some embodiments, L is a covalent bond.

[0266] In some embodiments, L is selected from C having 1-50 heteroatoms. 1-100 aliphatic and C 1-100 A heteroaliphatic divalent or optionally substituted straight-chain or branched group, wherein one or more methylene units in said group are optionally and independently substituted with: C 1-6 Alkylene, C 1-6 alkenyl group, divalent C with 1-5 heteroatoms 1-6 heteroaliphatic groups, , −Cy−, −C(R')2−, −O−, −S−, −S−S−, −N(R')−, −C(O)−, −C(S)−, −C(NR')−, −C(O)N(R')−, −C(O)C(R ')2N(R')−, −N(R')C(O)N(R')−, −N(R')C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R')−, −C(O)S−, −C(O )O−、−P(O)(OR')−、−P(O)(SR')−、−P(O)(R')−、−P(O)(NR')−、−P(S)(OR')−、−P(S)(SR')−、−P(S)(R')−、−P(S)(NR')−、−P(R')−、−P(OR')−、−P(SR')−、−P(NR')−、amino acid residues or −[(−O−C(R')2−C(R')2−) n In some embodiments, L is selected from C having 1-10 heteroatoms. 1-20 aliphatic and C 1-20 A heteroaliphatic divalent or optionally substituted straight-chain or branched group, wherein one or more methylene units in said group are optionally and independently substituted with: C 1-6 Alkylene, C 1-6 alkenyl group, divalent C with 1-5 heteroatoms 1-6 heteroaliphatic groups, , −Cy−, −C(R')2−, −O−, −S−, −S−S−, −N(R')−, −C(O)−, −C(S)−, −C(NR')−, −C(O)N(R')−, −C(O)C(R ')2N(R')−, −N(R')C(O)N(R')−, −N(R')C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R')−, −C(O)S−, −C(O )O−、−P(O)(OR')−、−P(O)(SR')−、−P(O)(R')−、−P(O)(NR')−、−P(S)(OR')−、−P(S)(SR')−、−P(S)(R')−、−P(S)(NR')−、−P(R')−、−P(OR')−、−P(SR')−、−P(NR')−、amino acid residues or −[(−O−C(R')2−C(R')2−) n In some embodiments, L is selected from C. 1-20 Aliphatic divalent or optionally substituted straight-chain or branched groups, wherein one or more methylene units of said group are optionally and independently replaced by: , −Cy−, −C(R')2−, −O−, −S−, −S−S−, −N(R')−, −C(O)−, −C(S)−, −C(NR')−, −C(O)N(R')−, −C(O)C(R ')2N(R')−, −N(R')C(O)N(R')−, −N(R')C(O)O−, −S(O)−, −S(O)2−, −S(O)2N(R')−, −C(O)S−, −C(O )O−、−P(O)(OR')−、−P(O)(SR')−、−P(O)(R')−、−P(O)(NR')−、−P(S)(OR')−、−P(S)(SR')−、−P(S)(R')−、−P(S)(NR')−、−P(R')−、−P(OR')−、−P(SR')−、−P(NR')−、amino acid residues or −[(−O−C(R')2−C(R')2−) n In some embodiments, L is a divalent or optionally substituted linear or branched C 1-20 Aliphatic, wherein one or more methylene units of said group are optionally and independently replaced by: -Cy−, -C(R')2−, -O−, -S−, -S−S−, -N(R')−, -C(O)−, -C(S)−, -C(NR')−, -C(O)N(R')−, -C(O)C(R')2N(R')−, -N(R')C(O)N(R')−, -N(R')C(O)O−, -S(O)−, -S(O)2−, -S(O)2N(R')−, -C(O)S−, -C(O)O−, amino acid residues or -[(−O−C(R')2−C(R')2−) n In some embodiments, L is a divalent or optionally substituted straight-chain or branched group C. 1-100 Aliphatic, wherein one or more methylene units of said group are optionally and independently replaced by: 、−Cy−、−C(R')2−、−O−、−N(R')−、−C(O)−、−C(S)−、−C(NR')−、−C(O)N(R')−、−C(O)C(R')2N(R')−、−N(R')C(O)N(R')−、−N(R')C(O)O−、−S(O)−、−S(O)2−、−S(O)2N(R')−、amino acid residues or −[(−O−C(R')2−C(R')2−) n In some embodiments, L is a divalent or optionally substituted linear or branched chain C. 1-50 An aliphatic group, wherein one or more methylene units of said group are optionally and independently substituted as described herein. In some embodiments, L is a divalent or optionally substituted straight-chain or branched C. 1-40 Aliphatic, wherein one or more methylene units of the group are optionally and independently substituted as described herein. In some embodiments, L is a divalent or optionally substituted straight-chain or branched C. 1-20 Aliphatic, wherein one or more methylene units of the group are optionally and independently substituted as described herein. In some embodiments, L is a divalent or optionally substituted straight-chain or branched C. 1-10 Aliphatic, wherein one or more methylene units of the group are optionally and independently substituted as described herein. In some embodiments, L is a divalent or optionally substituted straight-chain or branched C. 1-100 Alkylene, wherein one or more methylene units of the group are optionally and independently substituted as described herein. In some embodiments, L is a divalent or optionally substituted straight-chain or branched C. 1-50 Alkylene, wherein one or more methylene units of the group are optionally and independently substituted as described herein. In some embodiments, L is a divalent or optionally substituted straight-chain or branched C. 1-40Alkylene, wherein one or more methylene units of the group are optionally and independently substituted as described herein. In some embodiments, L is a divalent or optionally substituted straight-chain or branched C. 1-20 Alkylene, wherein one or more methylene units of the group are optionally and independently substituted as described herein. In some embodiments, L is a divalent or optionally substituted straight-chain or branched C. 1-10 Alkylene, wherein one or more methylene units of the group are optionally and independently replaced as described in this specification.

[0267] In some embodiments, the connector portions, such as L, L PM L RM Such as those containing acidic groups, for example, −S(O)2OH.

[0268] In some embodiments, L is or contains −[(−O−C(R')2−C(R')2−) n In some embodiments, L is or contains −[(−O−CH2−CH2−) n In some embodiments, L is −[(−CH2−CH2−O)6]−CH2−CH2−. In some embodiments, L is −[(−CH2−CH2−O)8]−CH2−CH2−. In some embodiments, −CH2−CH2−O− is bonded to the antibody-binding moiety at −CH2−. In some embodiments, −CH2−CH2−O− is bonded to the cell receptor-binding moiety at −CH2−. In some embodiments, L PM It is this type of L. In some embodiments, L RM It's this kind of L.

[0269] In some embodiments, the connector portion or L is or contains −(CH2CH2O)n−, wherein each −CH2− is independently and optionally substituted, and n is 1-20. In some embodiments, the connector portion or L is or contains −(CH2)n−O−(CH2CH2O)n−(CH2)n−, wherein each n is independently 1-10, and each −CH2− is independently and optionally substituted.

[0270] In some embodiments, the connector portion is trivalent or polyvalent. In some embodiments, the connector portion is L, where L is trivalent or polyvalent. In some embodiments, L is trivalent. In some embodiments, L is −CH2−N(−CH2−)−C(O)−.

[0271] In some embodiments, the connector portion, such as L, contains one or more amino acid residues or the like.

[0272] In some embodiments, the connector portion, such as L, LRM The reagent may contain or be a reactive group as described in this specification. In some embodiments, the reagent comprises an antibody-binding portion and a cell receptor-binding portion connected by a linker, the linker being or containing a reactive group. In some embodiments, the reactive group may react with lysine residues of the antibody in an aqueous buffer. In some embodiments, the reactive group is or contains −C(O)−O−. In some embodiments, the reactive group is or contains −C(O)−O−, wherein −O− is bonded to an optionally substituted aryl group. In some embodiments, the reactive group is or contains −C(O)−O−, wherein −O− is bonded to an aryl group substituted with one or more electron-withdrawing groups. In some embodiments, one or more or each electron-withdrawing group is independently selected from −NO2 and −F. In some embodiments, the aryl group has The structure, where R s It is a halogen, −NO2, −F, −L−R', −C(O)−L−R', −S(O)−L−R', −S(O)2−L−R', or −P(O)(−L−R')2. In some embodiments, the aryl group has The structure, where each R s Independently, it is a halogen, −NO2, −F, −L−R', −C(O)−L−R', −S(O)−L−R', −S(O)2−L−R', or −P(O)(−L−R')2. In some embodiments, the aryl group is... In some embodiments, the aryl group is In some embodiments, C1 binds to the -O- of -C(O)-O-. In some embodiments, the cell receptor binding portion is located on the -C(O)- side, and the antibody binding portion is located on the -O- side.

[0273] In some embodiments, the connector portion, such as L, L RM The reactive group includes a reactive group that reacts with a group of the antibody upon contact with the antibody, and optionally conjugates a cell receptor-binding portion or a portion containing −(Xaa)y− to the antibody via a linker. In some embodiments, the reactive group is or contains , wherein −C(O)− is optionally linked to the cell receptor-binding portion or the portion containing −(Xaa)y− via a linker. In some embodiments, the reactive group is or contains , wherein −C(O)− is optionally linked to the cell receptor binding portion or the portion containing −(Xaa)y− via a linker, and the other end of the reactive group is linked to the antibody binding portion.

[0274] In some embodiments, L is or comprises a bioorthogonal or enzymatic reaction product moiety. In some embodiments, L is or comprises an optionally substituted triazole moiety, which is optionally part of a bicyclic or polycyclic ring system. In some embodiments, L is or comprises LPXTG (SEQ ID NO: 74). In some embodiments, L is or comprises LPETG (SEQ ID NO: 75). In some embodiments, L is or comprises LPXT(G)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, L is or comprises LPET(G)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0275] In some embodiments, the connector portion (e.g., L, L) RM (etc.) do not have reactive groups. In some embodiments, the connector portion (e.g., L, L) RM (etc.) do not have reactive groups that readily react with proteins under aqueous conditions (e.g., physiological conditions) at a pH of approximately 6-9. In some embodiments, the connector portion (e.g., L, L) RM (etc.) do not have reactive groups that readily react with natural amino acid residues under aqueous conditions (e.g., physiological conditions) at a pH of about 6-9.

[0276] In some embodiments, the connector portion (e.g., L, L) RM (etc.) does not contain −S−, wherein neither of the two atoms bonded to −S− is S. In some embodiments, the linker portion (e.g., L, L) RM (etc.) does not contain −S−S−. In some embodiments, the connector portion (e.g., L, L) RM (etc.) does not contain −S− bonded to the β carbon of the carbonyl group or to a double or triple bond conjugated to the carbonyl group. In some embodiments, the connector portion, such as L, L RM etc., excluding In some embodiments, the connector portion, such as L, L... RM etc., excluding −S−.

[0277] In some embodiments, the agent does not contain a cleavable group that releases LG, except for one or more of optionally RG. In some embodiments, the agent does not contain -S−S−, acetal, or imine groups. In some embodiments, the agent does not contain -S−S−, acetal, or imine groups, except that the agent may have -S−S− formed by two amino acid residues. In some embodiments, the agent does not contain -S−S−, acetal, or imine groups, except that the agent may have -S−S− formed by cysteine ​​residues. In some embodiments, the agent does not contain -S−S−, acetal, or imine groups.

[0278] In some embodiments, L is a covalent bond. In some embodiments, L is a divalent, optionally substituted, straight or branched C. 1-100 An aliphatic group, wherein one or more methylene units in the group are optionally and independently substituted. In some embodiments, L is a divalent, optionally substituted, straight-chain or branched C. 6-100 An aryl aliphatic group, wherein one or more methylene units in the group are optionally and independently substituted. In some embodiments, L is a divalent, optionally substituted, straight-chain or branched C having 1 to 20 heteroatoms. 5-100 A heteroaryl aliphatic group, wherein one or more methylene units in the group are optionally and independently substituted. In some embodiments, L is a divalent, optionally substituted, straight-chain or branched C having 1 to 20 heteroatoms. 1-100 A heteroaliphatic group, wherein one or more methylene units in the group are optionally and independently replaced.

[0279] In some embodiments, the connector portion (e.g., L) is or comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) polyethylene glycol units. In some embodiments, the connector portion is or comprises −(CH2CH2O). n −, where n is as described in this specification. In some embodiments, one or more methylene units of L are independently −(CH2CH2O). n - Alternative.

[0280] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. In some embodiments, n is 11. In some embodiments, n is 12. In some embodiments, n is 13. In some embodiments, n is 14. In some embodiments, n is 15. In some embodiments, n is 16. In some embodiments, n is 17. In some embodiments, n is 18. In some embodiments, n is 19. In some embodiments, n is 20.

[0281] In some embodiments, the linker portion is or comprises one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues. In some embodiments, one or more methylene units of L are independently replaced by amino acid residues. In some embodiments, one or more methylene units of L are independently replaced by amino acid residues, wherein the amino acid residues belong to an amino acid of formula LNK101 or a salt thereof. In some embodiments, one or more methylene units of L are independently replaced by amino acid residues, wherein each amino acid residue independently has −N(R a1 )−L a1 −C(R a2 (R) a3 )−L a2 The structure of -CO− or its salt form.

[0282] In some embodiments, the connector portion includes one or more portions that can be used for connection with other portions, such as amino, carbonyl, etc. In some embodiments, the connector portion includes one or more −NR'−, wherein R' is as described in this specification. In some embodiments, −NR'− improves solubility. In some embodiments, −NR'− serves as a connection point with another portion. In some embodiments, R' is −H. In some embodiments, one or more methylene units of L are independently replaced by −NR'−, wherein R' is as described in this specification.

[0283] In some embodiments, the connector portion, such as L, contains a −C(O)− group, which can be used for connection with the portion. In some embodiments, one or more methylene units of L are independently replaced by −C(O)−.

[0284] In some embodiments, the connector portion, such as L, contains an −NR'− group, which can be used for connection with the portion. In some embodiments, one or more methylene units of L are independently replaced by −N(R')−.

[0285] In some embodiments, the connector portion, such as L, contains a −C(O)NR'− group, which can be used for connection with the portion. In some embodiments, one or more methylene units of L are independently replaced by −C(O)N(R')−.

[0286] In some embodiments, the connector portion, such as L, contains an −C(R')2− group. In some embodiments, one or more methylene units of L are independently replaced by −C(R')2−. In some embodiments, −C(R')2− is −CHR'−. In some embodiments, R' is −(CH2)2C(O)NH(CH2).11 COOH. In some embodiments, R' is −(CH2)2COOH. In some embodiments, R' is −COOH.

[0287] In some embodiments, the linker portion is or comprises one or more ring portions, for example, one or more methylene units of L are replaced by −Cy−. In some embodiments, the linker portion, such as L, comprises an aryl ring. In some embodiments, the linker portion (e.g., L) comprises a heteroaryl ring. In some embodiments, the linker portion, such as L, comprises an aliphatic ring. In some embodiments, the linker portion (e.g., L) comprises a heterocyclic ring. In some embodiments, the linker portion, such as L, comprises a polycyclic ring. In some embodiments, the ring in the linker portion, such as L, is 3- to 20-membered. In some embodiments, the ring is 5-membered. In some embodiments, the ring is 6-membered. In some embodiments, the ring in the linker is the product of a cycloaddition reaction (e.g., click chemistry and its variants) used to connect different portions.

[0288] In some embodiments, the connector portion (e.g., L) is or includes In some embodiments, the methylene unit of L is Alternative. In some embodiments, the methylene unit of L is replaced by −Cy−. In some embodiments, −Cy− is.

[0289] In some embodiments, the connector portion (e.g., L) is or contains −CO)y−. In some embodiments, L is or contains –[(CH2)nO]mCy[(CH2)nO]mNH, or L is –[(CH2)nO]mCy[(CH2)nO]mNHC(O)[(CH2)nO]mNH-, or L is –[(CH2)nO]mCy[(CH2)nO]m{NHC(O)[(CH2)nO]m}pNH-, wherein n, m, and p are independently selected from 1-20, 1-12, or 2-10 each time they appear. In some embodiments, each n is 2, m is independently selected from an integer from 2-10 each time it appears, or in some embodiments, m is independently selected from an integer from 2-6, and Cy is In some embodiments, the methylene unit of L is replaced by −Cy−. In some embodiments, −Cy− is In some embodiments, −Cy− is In some embodiments, −Cy− is .

[0290] In some embodiments, the connector portion (e.g., L) of the provided pharmaceutical agent includes In some embodiments, It is in this structure or In some embodiments, yes In some embodiments, yes .

[0291] In some embodiments, L is L 1 In some embodiments, L is L b .

[0292] In some embodiments, L RM It is a covalent bond. In some embodiments, L RM It is not a covalent bond. In some embodiments, L RM It is or contains −(CH2CH2O)n−. In some embodiments, L RM It is or contains −(CH2)n−O−(CH2CH2O)n−(CH2)n−, wherein each n is independently as described in this specification, and each −CH2− is independently optionally substituted. In some embodiments, L RM It is −(CH2)n−O−(CH2CH2O)n−(CH2)n−, where each n is independently as described in this specification, and each −CH2− is independently optionally substituted. In some embodiments, L RM It is −(CH2)2−O−(CH2CH2O)n−(CH2)2−, where n is as described in this specification, and each −CH2− is independently and optionally substituted. In some embodiments, L RM It is −(CH2)2−O−(CH2CH2O)n−(CH2)2−, where n is as described in this specification.

[0293] In some embodiments, L PM It is a covalent bond. In some embodiments, L PM It is not a covalent bond. In some embodiments, L PM It is or contains −(CH2CH2O)n−. In some embodiments, L PM It is or contains −(CH2)n−O−(CH2CH2O)n−(CH2)n−, wherein each n is independently as described in this specification, and each −CH2− is independently optionally substituted. In some embodiments, L PM It is −(CH2)n−O−(CH2CH2O)n−(CH2)n−, where each n is independently as described in this specification, and each −CH2− is independently optionally substituted. In some embodiments, L PMIt is −(CH2)2−O−(CH2CH2O)n−(CH2)2−, where n is as described in this specification, and each −CH2− is independently and optionally substituted. In some embodiments, L PM It is −(CH2)2−O−(CH2CH2O)n−(CH2)2−, where n is as described in this specification.

[0294] In some embodiments, L PM (For example, in the product of the first and second pharmaceutical agents) is or contains a reaction product portion formed by the first reactive portion and the second reactive portion.

[0295] In some embodiments, the connector portion (e.g., L in the product of the first and second agents) PM ) is or contains In some embodiments, the connector portion, such as L or a connector portion that may be L (e.g., L... RM L PM The methylene unit of (etc.) is replaced by −Cy−. In some embodiments, −Cy− is optionally substituted. In some embodiments, −Cy− is In some embodiments, L is –[(CH2)nO]mCH2Cy[(CH2)nO]m-. In some embodiments, −Cy− is In some embodiments, −Cy− is In some embodiments, −Cy− is .

[0296] Cell receptor binding portion

[0297] According to embodiments of the present invention, several receptor binding portions are described in: International Patent Publication WO2019 / 199621, published on October 17, 2019; International Patent Publication WO2019 / 199634, published on October 17, 2019; and International Patent Publication No. WO 2021 / 072246, each of which is incorporated herein by reference.

[0298] In one embodiment, the cell receptor binding portion may include a desialyl glycoprotein receptor (ASGPR) binding group connected to the linker portion via an amine group.

[0299] The amine group can be a primary alkylamine group or a secondary alkylamine group, each of which is optionally substituted with a C1-C3 alkyl group.

[0300] The cell receptor binding portion may include an ASGPR binding group according to the following chemical structure:

[0301] ,

[0302] [TBT101] or

[0303] ,

[0304] [TBT102]

[0305] Where X has a length of 1-4 atoms and contains O, S, N(R) N1 ) or C(R N1 (R) N1 ) group, such that:

[0306] When the length of X is one atom, X is O, S, N(R) N1 ) or C(R N1 (R) N1 ),

[0307] When the length of X is 2 atoms, at most one atom of X is O, S, or N (R). N1 ),

[0308] When the length of X is 3 or 4 atoms, no more than 2 atoms of X are O, S, or N (R). N1 );

[0309] Where R N1 It is H; or a C1-C3 alkyl group, which is optionally substituted with 1-3 halogroups;

[0310] R1 and R3 are independent of each other:

[0311] H; -(CH2) K OH; -(CH2) K OC1-C4 alkyl groups, optionally substituted with 1-3 halogenated groups; C1-C4 alkyl groups, optionally substituted with 1-3 halogenated groups; -(CH2) K -Vinyl; O-(CH2) K -Vinyl; -(CH2) K -Alynyl group; -(CH2) K -COOH;

[0312] -(CH2) K C(O)O-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups; OC(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups; -C(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups, or

[0313] R1 and R3 are independent of each other. Groups, optionally substituted with up to three halogenated groups; C1-C4 alkyl groups, each of which is optionally substituted with one to three halogenated groups or one or two hydroxyl groups; or O-C1-C4 alkyl groups, each of which is optionally substituted with one to three halogenated groups or one or two hydroxyl groups; and

[0314] K is an integer independently between 0 and 4, or

[0315] R1 and R3 are each independently groups based on the following chemical structures:

[0316] , where R 7 It is an O-C1-C4 alkyl group, optionally substituted with one to three halogenated groups, one or two hydroxyl groups, or R 7 yes

[0317] -NR N3 R N4 Group or; or

[0318] R1 and R3 are each independently groups based on the following structure:

[0319] Based on the following chemical structure , , , , or Group:

[0320] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,or or

[0321] R1 and R3 are independent of each other. Groups, in which It is a C3-C8 saturated carbon ring group;

[0322] R C It does not exist; it is H; a C1-C4 alkyl group, optionally substituted with 1-3 halogroups or 1-2 hydroxyl groups; or a group according to the following structure:

[0323]

[0324] R4, R5, and R6 are each independently H; halogenated (F, Cl, Br, I); CN; NR N1 R N2 ;-(CH2) K OH;

[0325] -(CH2) K OC1-C4 alkyl groups, optionally substituted with 1-3 halogenated groups; C1-C3 alkyl groups, optionally substituted with 1-3 halogenated groups; -O-C1-C3-alkyl groups, optionally substituted with 1-3 halogenated groups; -(CH2) K COOH;-(CH2) K C(O)O-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups; OC(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups; -C(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups, or

[0326] R C yes Groups, Groups,

[0327] Group or Group,

[0328] Where R N R N1 and R N2 Each is independently H; or a C1-C3 alkyl group, which is optionally substituted with one to three halogenated groups or one or two hydroxyl groups;

[0329] K is an integer independently between 0 and 4;

[0330] K' is an integer from 1 to 4;

[0331] R N3It is H; or a C1–C3 alkyl group, optionally substituted with 1–3 halogenated groups or 1 or 2 hydroxyl groups; and

[0332] R N4 It is H; C1-C3 alkyl, which is optionally substituted with 1-3 halogenated groups or 1 or 2 hydroxyl groups, or R N4 It is a functional group, wherein K is preferably 1;

[0333] It is a linker group that contains at least one IgG4 binding moiety and optionally links at least one IgG4 binding moiety to a cell receptor binding moiety via the linker moiety, or

[0334] It is a linker group having at least one or more functional groups, which can be used to covalently bond the linker group to at least one IgG4 binding moiety or optionally a linker moiety;

[0335] R2 is Group, wherein R N1 And K is the same as above;

[0336] R AM It is H; C1-C4 alkyl, optionally substituted with up to 3 halogenated groups and one or two hydroxyl groups; -(CH2) K COOH group; -(CH2) K C(O)O-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups; OC(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated F groups; -C(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups; -(CH2) K -NR N3 R N4 Group, wherein R N3 It is H; or a C1-C3 alkyl group, optionally substituted with 1-3 halogenated groups or 1 or 2 hydroxyl groups; and

[0337] R N4 It is H; C1-C3 alkyl, optionally substituted with 1-3 halogenated groups or 1 or 2 hydroxyl groups; or a group, or

[0338] R2 is Group,

[0339] Where R TA It is H; CN; NR N1 R N2 ;-(CH2) K OH; -(CH2) KOC1-C4 alkyl, which is optionally substituted with 1-3 halogenated groups; C1-C4 alkyl, which is optionally substituted with 1-3 halogenated groups;

[0340] -(CH2) K COOH;-(CH2) K C(O)O-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups; OC(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups; -C(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups, or

[0341] R TA It is C3-C 10 The aryl group or a tri- to deca-aryl group containing up to five heteroaryl atoms, each of which is optionally substituted by up to three (preferably one) of the following: CN; NR N1 R N2 ;-(CH2) K OH; -(CH2) K OC1-C4 alkyl groups, optionally substituted with 1-3 halogenated groups; C1-C3 alkyl groups, optionally substituted with 1-3 halogenated groups or 1 or 2 hydroxyl groups; -O-C1-C3 alkyl groups, optionally substituted with 1-3 halogenated groups; -(CH2) K COOH;-(CH2) K C(O)O-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups; OC(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups; or -(CH2) K C(O)-C1-C4 alkyl, which is optionally substituted with 1-3 halogenated groups, or

[0342] R TA yes Group; Group; Group; Group; A group, which is optionally substituted with up to three C1-C3 alkyl groups optionally substituted with up to three halogenated groups, or

[0343] R TA It is a functional group.

[0344] Where R N R N1 and R N2 Each is independently H; or a C1-C3 alkyl group, optionally substituted with one to three halogen groups or one or two hydroxyl groups, and each -(CH2) KThe group is optionally replaced by 1 to 4, preferably 1 or 2, C1-C3 alkyl groups, which are optionally replaced by 1 to 3 fluorinated groups or 1 to 2 hydroxyl groups;

[0345] And K is independently 0-4.

[0346] The cell receptor binding portion may include an ASGPR binding group according to the following chemical structure:

[0347] or

[0348] Where X has a length of 1-4 atoms and contains O, S, N(R) N1 ) or C(R N1 (R) N1 ) group, such that:

[0349] When the length of X is one atom, X is O, S, N(R) N1 ) or C(R N1 (R) N1 ),

[0350] When the length of X is 2 atoms, at most one atom of X is O, S, or N (R). N1 ),

[0351] When the length of X is 3 or 4 atoms, no more than 2 atoms of X are O, S, or N (R). N1 );

[0352] Each R N1 Independently H; or C1-C3 alkyl, optionally substituted with 1-3 halogenated groups, preferably F-substituted (R N1 Preferably H or methyl, more typically H);

[0353] R1 and R3 are each independently H; -(CH2) K OH; -(CH2) K OC1-C4 alkyl groups, optionally substituted with 1-3 halogenated (F, Cl, Br, I, preferably F) groups; C1-C4 alkyl groups, optionally substituted with 1-3 halogenated (F, Cl, Br, I, preferably F) groups; -(CH2) K Vinyl; O-(CH2) K vinyl;

[0354] -(CH2) K Alkyne group; -(CH2) K COOH;-(CH2) KC(O)O-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups, preferably F-substituted groups; OC(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups, preferably F-substituted groups; -C(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups, preferably F-substituted groups, or

[0355] R1 and R3 are independent of each other. A group, optionally substituted with up to three (preferably one) halogenated groups (preferably F); a C1-C4 alkyl group, each optionally substituted with one to three halogenated groups, preferably F or one or two hydroxyl groups; or

[0356] O-C1-C4 alkyl groups, each of which is optionally substituted with one to three halogenated groups, preferably F or one or two hydroxyl groups, and K is independently 0-4 (0, 1, 2, 3 or 4), or

[0357] R1 and R3 are each independently groups based on the following chemical structures:

[0358] , where R 7 It is an O-C1-C4 alkyl group, optionally substituted with one to three halogenated groups, preferably F and one or two hydroxyl groups, or R 7 yes

[0359] -NR N3 R N4 Group or ,or

[0360] R1 and R3 are each independently groups based on the following structure:

[0361] , , , , or

[0362] Based on the following chemical structure Group:

[0363] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or ,

[0364] Or R1 and R3 are independent of each other. Groups, in which It is a C3-C8 saturated carbon ring group;

[0365] R C It does not exist; it is H; C1-C4 alkyl, optionally substituted with 1-3 halo (preferably fluorinated) groups or 1-2 hydroxyl groups; or groups according to the following structures:

[0366]

[0367] R4, R5, and R6 are each independently H; halogenated (F, Cl, Br, I); CN; NR N1 R N2 ;

[0368] -(CH2) K OH; -(CH2) K OC1-C4 alkyl groups, optionally substituted with 1-3 halogenated (F, Cl, Br, I, preferably F) groups; C1-C3 alkyl groups, optionally substituted with 1-3 halogenated (F, Cl, Br, I, preferably F) groups; -O-C1-C3-alkyl groups, optionally substituted with 1-3 halogenated, preferably F groups; -(CH2) K COOH;-(CH2) K C(O)O-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups, preferably F-substituted groups; OC(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups, preferably F-substituted groups; -C(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups, preferably F-substituted groups, or

[0369] R C yes Groups, Groups,

[0370] Group or Group,

[0371] Where R N R N1 and R N2 Each is independently H, or C1-C3 alkyl, which is optionally substituted with one to three halogenated groups (preferably F) or one or two hydroxyl groups;

[0372] K is independently 0-4 (0, 1, 2, 3 or 4), preferably 0 or 1;

[0373] K' is 1-4, preferably 1;

[0374] R N3 It is H; or a C1-C3 alkyl group, optionally substituted with 1-3 halogenated groups, preferably F or 1 or 2 hydroxyl groups; and

[0375] R N4 It is H; C1-C3 alkyl, which is optionally substituted with 1-3 halogenated groups, preferably F or 1 or 2 hydroxyl groups, or R N4 It is a functional group, wherein K is preferably 1;

[0376] It is a linker group that contains at least one IgG4 binding moiety and optionally links at least one IgG4 binding moiety to a cell receptor binding moiety via the linker moiety, or

[0377] It is a linker group having at least one or more functional groups, which can be used to covalently bond the linker group to at least one IgG4 binding moiety or optionally a linker moiety;

[0378] R2 is Group, wherein R N1 And K is the same as above;

[0379] R AM It is H; C1-C4 alkyl, optionally substituted with up to 3 halogenated groups (preferably F) and one or two hydroxyl groups; -(CH2) K COOH group; -(CH2) K C(O)O-C1-C4 alkyl, which is optionally substituted with 1-3 halogroups, preferably F groups;

[0380] OC(O)-C1-C4 alkyl, optionally substituted with 1-3 halogens, preferably with an F group; -C(O)-C1-C4 alkyl, optionally substituted with 1-3 halogens, preferably with an F group; -(CH2) K -NR N3 R N4 Group, wherein RN3 It is H; or a C1-C3 alkyl group, optionally substituted with 1-3 halogenated groups, preferably F or 1 or 2 hydroxyl groups, or

[0381] R2 is Group,

[0382] Where R TA It is H; CN; NR N1 R N2 ;-(CH2) K OH; -(CH2) K OC1-C4 alkyl groups, optionally substituted with 1-3 halogenated (F, Cl, Br, I, preferably F) groups; C1-C4 alkyl groups, optionally substituted with 1-3 halogenated (F, Cl, Br, I, preferably F) groups; -(CH2) K COOH;-(CH2) K C(O)O-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups, preferably F-substituted; OC(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups, preferably F-substituted; -C(O)-C1-C4 alkyl, optionally substituted with 1-3 halogenated groups, preferably F-substituted; or R TA It is C3-C 10 The aryl group or a tri- to deca-aryl group containing up to five heteroaryl atoms, each of which is optionally substituted by up to three (preferably one) of the following: CN; NR N1 R N2 ;-(CH2) K OH; -(CH2) K OC1-C4 alkyl groups, optionally substituted with 1-3 halogenated (F, Cl, Br, I, preferably F) groups; C1-C3 alkyl groups, optionally substituted with 1-3 halogenated (F, Cl, Br, I, preferably F) groups or 1 or 2 hydroxyl groups; -O-C1-C3-alkyl groups, optionally substituted with 1-3 halogenated, preferably F groups; -(CH2) K COOH;-(CH2) K C(O)O-C1-C4 alkyl, optionally substituted with 1-3 halogens, preferably with an F group; OC(O)-C1-C4 alkyl, optionally substituted with 1-3 halogens, preferably with an F group; or -(CH2) K C(O)-C1-C4 alkyl, optionally substituted with 1-3 halogens, preferably with an F group, or

[0383] R TA yes Groups, Groups, Group or Group, or

[0384] R TA yes The group, optionally substituted by up to three, preferably one C1-C3 alkyl group, wherein the alkyl group is optionally substituted by up to three halogenated (preferably F) groups, or

[0385] R TA yes Group,

[0386] Where R N R N1 and R N2 Each is independently H, or a C1-C3 alkyl group, optionally substituted with one to three halogenated groups (preferably F) or one or two hydroxyl groups; and

[0387] Each of the -(CH2) K The group is optionally substituted by 1 to 4, preferably 1 or 2 C1-C3 alkyl groups, which are optionally substituted by 1 to 3 fluorinated groups or 1 to 2 hydroxyl groups;

[0388] And K is independently 0-4 (0, 1, 2, 3 or 4), preferably 0 or 1; or

[0389] Its pharmaceutically acceptable salts, stereoisomers, solvates, or polymorphs.

[0390] In one embodiment,

[0391] When the length of X is 2 atoms, X is -OC(R) N1 (R) N1 ),

[0392] C(R N1 (R) N1 )-O-、-SC(R N1 (R) N1 ), C(R N1 (R) N1 )-S-、N(R N1 )-C(R N1 (R) N1 ),

[0393] C(R N1 (R) N1 )-N(R N1 ) or C(R N1 (R) N1 )-C(R N1 (R) N1 ),

[0394] When the length of X is 3 atoms, X is -O-C(R N1 )(R N1 )-C(R N1 )(R N1 ), C(R N1 )(R N1 )-O-C(R N1 )(R N1 )-,

[0395] -O-C(R N1 )(R N1 )-O-, -O-C(R N1 )(R N1 )-S-, -O-C(R N1 )(R N1 )-N(R N1 )-,

[0396] -S-C(R N1 )(R N1 )-C(R N1 )(R N1 ), C(R N1 )(R N1 )-S-C(R N1 )(R N1 )-, C(R N1 )(R N1 )-C(R N1 )(R N1 )-S, -S-C(R N1 )(R N1 )-S-, -S-C(R N1 )(R N1 )-O-, -S-C(R N1 )(R N1 )-N(R N1 )-, N(R N1 )-C(R N1 )(R N1 )-C(R N1 )(R N1 ), C(R N1 )(R N1 )-N(R N1 )-C(R N1 )(R N1 ), C(R N1 )(R N1 )-C(R N1 )(R N1 )-N(R N1 ), N(R N1 )-C(R N1 )(R N1)-N(R N1 ) or C(R N1 (R) N1 )-C(R N1 (R) N1 )-C(R N1 (R) N1 ),and

[0397] When the length of X is 4 atoms, X is -OC(R) N1 (R) N1 )-C(R N1 (R) N1 )-C(R N1 (R) N1 ), C(R N1 (R) N1 )-OC(R N1 (R) N1 )-(R N1 (R) N1 )-、-OC(R N1 (R) N1 )-OC(R N1 (R) N1 )-、-SC(R N1 (R) N1 )-C(R N1 (R) N1 )- C(R N1 (R) N1 )-、C(R N1 (R) N1 )-SC(R N1 (R) N1 )-C(R N1 (R) N1 )-、C(R N1 (R) N1 )-(R N1 (R) N1 )-SC(R N1 (R) N1 )-、-SC(R N1 (R) N1 )-SC(R N1 (R) N1 )-、N(R N1 )-C(R N1 (R) N1 )-C(R N1 (R) N1 )- C(R N1 (R) N1 )-、C(R N1 (R) N1 )-N(RN1 )-C(R N1 (R) N1 )-C(R N1 (R) N1 ), C(R N1 (R) N1 )-C(R N1 (R) N1 )- N(R N1 ), N(R N1 )-C(R N1 (R) N1 )-N(R N1 ) or C(R N1 (R) N1 )-C(R N1 (R) N1 )- C(R N1 (R) N1 ),

[0398] Where R N1 Same as described above.

[0399] In one embodiment, X is OCH2 or CH2O, and R N1 It is H.

[0400] The cell receptor binding site can have the following structure:

[0401] or ;

[0402] Where R A It is a C1-C3 alkyl group, which is optionally substituted with 1-5 halogenated (preferably fluorinated) groups (preferably R...). A (It is methyl or ethyl, which may optionally be substituted with 1-3 fluorinated groups);

[0403] Z A It is -(CH2) IM -O-(CH2) IM S-(CH2) IM NR M -(CH2) IM ;C(O)-(CH2) IM -; containing 1 to 8, preferably 1 to 4, ethylene glycol residues of a PEG group; or -C(O)(CH2). IM NR M Groups (preferably containing PEG with groups comprising 1 to 8 ethylene glycol residues, preferably 2 to 4 ethylene glycol residues), wherein IM and R M Same as above; and

[0404] ZB It does not exist; it is (CH2). IM C(O)-(CH2) IM -or C(O)-(CH2) IM -NR M IM and R M Same as above.

[0405] In one embodiment, R A It can be methyl or ethyl, which may optionally be substituted with 1 to 3 fluorinated groups.

[0406] In one embodiment, Z A It can be a PEG group containing 1 to 4 ethylene glycol residues.

[0407] In one embodiment, the methyl or ethyl group may be substituted with 1-3 fluorinated groups.

[0408] In one embodiment, the ASGPR binding group may be N-acetyl-D-galactosamine.

[0409] In some embodiments, the cell receptor binding portion may be selected from the following compounds:

[0410]

[0411] Pharmaceutical compositions; pharmaceutically acceptable excipients

[0412] Formulas suitable for parenteral administration, such as those administered via intra-articular, intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injectable solutions containing antioxidants, buffers, and antibacterial agents, and solutes that make the formula isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In practice, the compositions can be administered via intravenous infusion, oral administration, topical administration, intraperitoneal administration, intravesical administration, or intrathecal administration. Parenteral, oral, and intravenous administration are methods of administration. Formulas of the compounds may be present in single-dose or multi-dose sealed containers, such as ampoules and vials.

[0413] Solutions or suspensions intended for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for adjusting osmotic pressure, such as sodium chloride or glucose. The pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0414] Methods for preparing pharmaceuticals .

[0415] The pharmaceutical agents described herein can be prepared or isolated according to this specification by synthetic and / or semi-synthetic or recombinant methods. In some embodiments, peptide agents, such as cell receptor-binding partial peptide agents, can be prepared using a biological expression system. In some embodiments, the provided pharmaceutical agents are synthetically prepared. In some embodiments, the provided pharmaceutical agents are prepared using certain techniques described in International Patent Publication WO 2019 / 023501, the entire contents of which are incorporated herein by reference.

[0416] Several techniques, such as those used to prepare antibody-drug conjugates, can be used to prepare MATE agents. In many of these techniques, the conjugation is not selective for amino acid residue sites, and the product composition typically has several different types of agents that may differ from one another in the number of target-binding moieties and / or conjugation sites. In some embodiments, the present invention provides techniques for selective conjugation to target-binding moieties at amino acid residue sites.

[0417] In some embodiments, the present invention provides a synthesis method, the method comprising the following steps:

[0418] A first agent comprising a cell receptor-binding portion optionally linked to a first reactive group via a first connector is contacted with a second agent comprising an antibody portion optionally linked to a second reactive group via a second connector, wherein the first reactive group reacts with the second reactive group; and

[0419] The product agent is formed comprising a cell receptor-binding portion and an antibody-binding portion optionally via a linker.

[0420] In some embodiments, the present invention provides a synthesis method, the method comprising the following steps:

[0421] A first composition comprising multiple first agents is contacted with a second composition comprising multiple second agents, each of the multiple first agents independently comprising a cell receptor binding portion optionally linked to a first reactive group via a first connector portion, and each of the multiple second agents independently comprising an antibody portion optionally linked to a second reactive group via a second connector portion.

[0422] The product composition is formed comprising multiple product agents, each of which independently comprises a cell receptor binding portion and an antibody binding portion optionally connected via a linker.

[0423] In some embodiments, LG is or includes an antibody-binding portion as described in this specification, and a connector connecting the antibody-binding portion and RG.

[0424] In some embodiments, L LG2 It is −N(R')−. In some embodiments, L LG2 It is −N(R)−. In some embodiments, L LG2 It is −NH−.

[0425] In some embodiments, L LG2 It is a divalent C that can be substituted by any choice. 1-6 Aliphatic. In some embodiments, L LG2 It is −CH2−. In some embodiments, L LG2 It is −CH2NH−. In some embodiments, L LG2 It is −CH2NH−C(O)−. In some embodiments, L LG2 It is −CH2NH−C(O)−CH2−.

[0426] In some embodiments, L LG3 It may contain, or optionally substituted, aryl rings. In some embodiments, L LG3 It is or contains an optionally substituted phenyl ring. In some embodiments, L LG3It is a benzene ring substituted with one or more electron-withdrawing groups. As will be understood by one of ordinary skill in the biomedical field, several electron-withdrawing groups are known in the biomedical field and can be used according to this specification. In some embodiments, the electron-withdrawing group is a halogen. In some embodiments, the electron-withdrawing group is −F. In some embodiments, the electron-withdrawing group is −Cl. In some embodiments, it is −Br. In some embodiments, it is −I. In some embodiments, the electron-withdrawing group comprises an X=Y double bond, wherein X is bonded to a group in which the electron-withdrawing group is a substituent, and at least one of X and Y is a heteroatom. In some embodiments, X is a heteroatom. In some embodiments, Y is a heteroatom. In some embodiments, each of X and Y is a heteroatom independently. In some embodiments, Y is O. In some embodiments, Y is S. In some embodiments, X is C. In some embodiments, X is N. In some embodiments, X is P. In some embodiments, X is S. In some embodiments, X=Y is C=O. In some embodiments, X=Y is N=O. In some embodiments, X=Y is S=O. In some embodiments, X=Y is P=O. In some embodiments, the electron-withdrawing group is −C(O)−L−R'. In some embodiments, the electron-withdrawing group is −C(O)−R'. In some embodiments, it is −NO2. In some embodiments, it is −S(O)−L−R'. In some embodiments, it is −S(O)−R'. In some embodiments, it is −S(O)2−L−R'. In some embodiments, it is −S(O)2−O−R'. In some embodiments, it is −S(O)2−N(R')2. In some embodiments, it is −P(O)(−L−R')2. In some embodiments, it is −P(O)(R')2. In some embodiments, it is −P(O)(OR')2. In some embodiments, it is −P(O)[N(R')2]2.

[0427] In some embodiments, L LG3 It is −L LG3a -L LG3b -, where L LG3a It is a covalent bond or −C(O)O−CH2−, where −CH2− is optionally substituted, and L LG3b It is an optionally substituted aryl ring. In some embodiments, L LG3a Bond to L LG2 And L LG3b Bond to L LG4 .

[0428] In some embodiments, L LG3a It is a covalent bond. In some embodiments, L LG3a It is −C(O)O−CH2−, where −CH2− is optionally substituted. In some embodiments, LLG3a It is −C(O)O−CH2−, where −CH2− is substituted. In some embodiments, L LG3a It is −C(O)O−CH2−, where −CH2− is unsubstituted.

[0429] In some embodiments, the first group, the antibody-binding portion, and / or LG serve as having R LG -L LG1 -L LG2 Compounds with an −H structure or a portion of their salts are released.

[0430] In some embodiments, L LG4 It is a covalent bond. In some embodiments, L LG4 It is not a covalent bond. In some embodiments, L LG4 It is −O−. In some embodiments, L LG4 It is −N(R')−. In some embodiments, L LG4 It is −NH−. In some embodiments, L LG4 It is −N(CH3)−. In some embodiments, L LG4 It is −N(R')−, and L LG3 It is −O−. In some embodiments, R' is optionally substituted C. 1−6 Alkyl group. In some embodiments, L LG4 It is −S−.

[0431] In some embodiments, R LG It may contain an antibody-binding portion. In some embodiments, R LG It may contain a protein-binding moiety. In some embodiments, R LG It may contain an antibody-binding portion. In some embodiments, R LG It is the antibody-binding portion. In some embodiments, R LG It is the protein-binding portion. In some embodiments, R LG It is the antibody-binding part.

[0432] In some embodiments, R LG It is ABT101, R c −(Xaa)z−, nucleic acid moieties, or small molecule moieties. In some embodiments, R LG It is or includes ABT101. In some embodiments, R LG It is or contains Rc−(Xaa)z−. In some embodiments, R LG It may contain small molecule portions. In some embodiments, R LG It is or contains peptides. In some embodiments, R LG It may contain nucleic acid agents. In some embodiments, R LGIt may contain an aptamer. In some embodiments, the antibody-binding portion is or contains ABT101. In some embodiments, the protein-binding portion is or contains ABT101. In some embodiments, the antibody-binding portion is or contains ABT101. In some embodiments, the antibody-binding portion is or contains Rc−(Xaa)z−. In some embodiments, the protein-binding portion is or contains Rc−(Xaa)z−. In some embodiments, the antibody-binding portion is or contains Rc−(Xaa)z−.

[0433] In some embodiments, the target-binding portion may optionally be conjugated to the antibody portion via a connector portion using the technique described in U.S. Patent Publication 2020 / 0190165.

[0434] In some embodiments, where a particular protecting group (PG), leaving group (LG), or transformation condition is described, those skilled in the art will recognize that other protecting groups, leaving groups, and transformation conditions are also suitable and expected. Such groups and transformations are described in Smith & March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (John Wiley & Sons, 2001), Larock, Comprehensive Organic Transformations, 2nd Edition (John Wiley & Sons, 1999), and Greene & Wuts, Protecting Groups in Organic Synthesis, 3rd Edition (John Wiley & Sons, 1999), the entire contents of each of which are hereby incorporated by reference in this specification.

[0435] In some embodiments, the leaving group includes, but is not limited to, halogens (e.g., fluorides, chlorides, bromides, iodides), sulfonates (e.g., methanesulfonates, toluenesulfonates, benzenesulfonates, p-bromobenzenesulfonates, nitrobenzenesulfonates, trifluoromethanesulfonates), diazo groups, etc.

[0436] In some embodiments, the oxygen protecting group includes a carbonyl protecting group, a hydroxyl protecting group, etc. Hydroxyl protecting groups are well known in the biomedical field and include those described in Greene & Wuts, Protecting Groups in Organic Synthesis, 3rd Edition (John Wiley & Sons, 1999), the entire contents of which are incorporated herein by reference. Examples of suitable hydroxyl protecting groups include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, aryl alkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formate esters, acetate esters, carbonate esters, and sulfonate esters. Specific examples include formate esters, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxovalerate, 4,4-(ethylenedithio)valerate, neovalerate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate esters, p-benzyl benzoate, 2,4,6-trimethylbenzoate, carbonates such as methyl ester, 9-fluorenylmethyl ester, ethyl ester, 2,2,2-trichloroethyl ester, 2-(trimethylsilyl)ethyl ester, 2-(phenylsulfonyl)ethyl ester, vinyl ester, allyl ester, and p-nitrobenzoate. Examples of such silyl ethers include trimethylsilyl, triethylsilane, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilane, and other trialkylsilyl ethers. Alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, triphenylmethyl, tert-butyl, allyl, and allyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl ether, methyl thiomethyl ether, (2-methoxyethoxy)methyl ether, benzyloxymethyl ether, β-(trimethylsilyl)ethoxymethyl ether, and tetrahydropyran ether. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, and 2-pyridinemethyl and 4-pyridinemethyl.

[0437] Amino protecting groups are well-known in the biomedical field and include those described in Greene & Wuts, Protecting Groups in Organic Synthesis, 3rd Edition (John Wiley & Sons, 1999), the entire contents of which are incorporated herein by reference. Suitable amino protecting groups include, but are not limited to, aralkylamines, carbamates, cyclic imides, allylamines, amides, etc. Examples of such groups include tert-butoxycarbonyl (BOC), ethoxycarbonyl, methoxycarbonyl, trichloroethoxycarbonyl, allyloxycarbonyl (Alloc), benzyloxycarbonyl (CBZ), allyl, phthalimide, benzyl (Bn), fluorenylmethylcarbonyl (Fmoc), formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, phenylacetyl, trifluoroacetyl, benzoyl, etc.

[0438] Those skilled in the biomedical field know that the provided pharmaceutical agents can have one or more stereocenters and can exist as a mixture of racemic or diastereomers. Those skilled in the biomedical field also know that numerous methods exist in the biomedical field for isolating isomers to obtain stereoenriched or stereopure isomers of those compounds, including but not limited to high-performance liquid chromatography, chiral high-performance liquid chromatography, stepwise crystallization of diastereomeric salts, kinetic enzymatic resolution (e.g., by fungal, bacterial, or animal-derived lipases or esterases), and the formation of covalent diastereomeric derivatives using enantiomeric enrichment reagents.

[0439] Those skilled in the biomedical field will know that several functional groups present in the compounds of this specification, such as aliphatic groups, alcohols, carboxylic acids, esters, amides, aldehydes, halogens, and nitriles, can be interconverted using techniques well known in the biomedical field, including but not limited to reduction, oxidation, esterification, hydrolysis, partial oxidation, partial reduction, halogenation, dehydration, partial hydration, and hydration. See Smith & March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (John Wiley & Sons, 2001), the entire contents of which are incorporated herein by reference. Such interconversions may require one or more of the above techniques, and some methods for synthesizing the compounds of this specification are described below in the illustrations.

[0440] Those skilled in the art of biomedicine will know that reaction couplers are typically in contact with each other under conditions and for a time sufficient to produce the desired results, such as forming a product agent and its composition to the desired degree. Many reaction conditions / reaction times can be evaluated and used if they are suitable for the intended purpose according to this specification; some such conditions, reaction times, evaluations, etc., are described in this specification.

[0441] In some embodiments, the formed agent, such as the product MATE agent, has a structure of formula AGN101 or AGN102 or a salt thereof. In some embodiments, the cell receptor binding portion in the product agent (e.g., the MATE agent) is the same as the cell receptor binding portion in the reaction partner used to prepare the product agent (e.g., a first agent containing the cell receptor binding portion). In some embodiments, the antibody portion in the product agent (e.g., the MATE agent) is the same as the antibody portion in the reaction partner used to prepare the product agent (e.g., a second agent containing the antibody portion).

[0442] In some embodiments, a linker portion or a portion thereof connected to the target binding portion and / or the antibody portion may be available from a reaction coupler (e.g., L of formula AGN301). RM Transfer of (or its salt). In some embodiments, the connector portion (which may be referred to as L) in the product agent is transferred. PM For example, L in formula AGN101 or AGN102 is or includes a linker portion in the reactive coupler, such as a linker portion between a reactive group and a cell receptor binding portion, for example, L. RM In some embodiments, L PM Is or contains L RM In some embodiments, L PM It is −L RM -L RG2 -. In some embodiments, L RG2 It is −C(O)−. In some embodiments, L RG2 It is −C(O)− and is bonded to −NH− of the target drug moiety, such as −NH− in the side chain of a lysine residue of the protein moiety, in some embodiments of which the protein moiety is an antibody moiety.

[0443] The reaction coupler (e.g., a compound of formula AGN301 or a salt thereof) rarely contains a portion that can react with the reactive group under conditions where the reactive group reacts with the target drug. In some embodiments, the reaction between such portions and the reactive group is significantly slower and / or less efficient than the reaction between the reactive group and the target drug, provided that some portions of the reaction coupler can react with the reactive group under conditions where the reactive group reacts with the target drug. In some embodiments, the reaction between such portions and the reactive group does not significantly reduce the amount, efficiency, yield, rate, and / or conversion of the reaction between the reactive group and the target drug, for example, by no more than about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc. In some embodiments, the reactive group (e.g., an ester group, an activated carboxylic acid derivative, etc.) reacts with the amino group (e.g., an -NH2 group) of the target drug (e.g., a protein drug, such as an antibody drug). In some embodiments, the reaction coupler (e.g., a compound of formula AGN301 or a salt thereof) does not have an amine group. In some embodiments, compounds of formula AGN301 or salts thereof (or portions thereof, such as R) LG L LG L LG1 L LG2 L LG3 L LG4 L RG1 L RG2 L RM (and / or MOI) do not have an amine group. In some embodiments, they do not have a primary amine group (−NH2). In some embodiments, they do not have −CH2NH2. In some embodiments, they do not have −CH2CH2NH2. In some embodiments, they do not have −CH2CH2CH2NH2. In some embodiments, they do not have −CH2CH2CH2CH2NH2. In some embodiments, the amine group (e.g., a primary amine group) is capped, for example by introducing an acyl group (e.g., R−C(O)−, such as an acetyl group to form an amide group) to prevent or reduce undesirable reactions.

[0444] In some embodiments, the reaction is carried out in a buffer system. In some embodiments, the buffer system of this disclosure maintains the structure and / or function of the target drug, cell receptor binding moiety, etc. In some embodiments, the buffer is a phosphate buffer. In some embodiments, the buffer is a PBS buffer. In some embodiments, the buffer is a borate buffer. In some embodiments, the buffer of this specification provides and optionally maintains a certain pH value or range. In some embodiments, a useful pH is about 7-9, such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 9.0, etc. In some embodiments, the pH is 7.4. In some embodiments, the pH is 7.5. In some embodiments, the pH is 7.8. In some embodiments, the pH is 8.0. In some embodiments, the pH is 8.2. In some embodiments, the pH is 8.3.

[0445] The provided technology offers several advantages. In some embodiments, the connection of the cell receptor-binding moiety in the reaction coupler (e.g., a compound containing a reactive group located between the antibody-binding moiety and the cell receptor-binding moiety, such as a composition of substance of formula AGN301 or a salt thereof) to an agent containing the antibody moiety (e.g., a second agent, such as an antibody agent), and the release of the antibody-binding moiety in the provided reaction coupler, can be achieved in a single reaction and / or in a single pot. Therefore, in many embodiments, no separate reaction / step is performed to remove the antibody-binding moiety. Those skilled in the biomedical field will recognize that by performing the connection of the cell receptor-binding moiety and the release of the antibody-binding moiety in a single reaction / operation, the provided technology avoids the separate step of antibody-binding moiety removal and can improve overall efficiency (e.g., by simplifying operations, increasing overall yield, etc.), reduce manufacturing costs, and improve product purity (e.g., by avoiding exposure to antibody-binding moiety removal conditions), which typically involve one or more of conditions such as reduction, oxidation, hydrolysis (e.g., hydrolysis of ester groups), and may impair its target agent moiety (e.g., for protein agent moieties, protein amino acid residues, overall structure, and / or post-translational modifications, such as antibody glycans). In some embodiments, the provided techniques may provide improved efficiency (e.g., in terms of reaction rate and / or conversion percentage), increased yield, increased purity / homogeneity, and / or enhanced selectivity, particularly compared to reference techniques that use reaction couplers without antibody-binding moieties, without introducing an antibody-binding moieties removal step, e.g., removing the antibody-binding moieties in the same step as conjugating cell receptor-binding moieties.

[0446] In some embodiments, the present invention provides a product of the provided process that has a low level of damage to the antibody portion compared to a process comprising steps performed to remove the antibody-binding portion rather than for substantial conjugation of the portion of interest (e.g., target-binding portion). In some embodiments, the provided product pharmaceutical composition has high homogeneity (e.g., regarding the number of cell receptor-binding portions of each antibody portion and / or the position of amino acid residues in the antibody portion conjugated with the portion of interest) compared to a reference product composition (e.g., from a technique that does not use the antibody-binding portion, or a technique that uses additional steps to remove the antibody-binding portion (e.g., without using the described reactive coupler containing a reactive group located between the antibody-binding portion and the cell receptor-binding portion)).

[0447] In some embodiments, the present invention provides a product pharmaceutical agent comprising an antibody portion, a cell receptor binding portion, and optionally a connector portion linking the antibody binding portion and the cell receptor binding portion. In some embodiments, the present invention provides compositions of such pharmaceutical agents.

[0448] GN3 sorting enzyme reagent The basic scheme for sorting enzyme conjugation includes an ASPGR binding agent for the sorting enzyme and a C-terminal sorting enzyme tag (LPETGG) (GN3 / linker) for conjugation.

[0449] GN3-maleimide reagent (Maleimide-GN3 / connector), with C-terminal Cys for fusion.

[0450] MATES

[0451] As used herein, the term "MATE" refers to Multimodal Antibody Therapy Enhancer, a next-generation antibody conjugation technology capable of site-specific pairing with therapeutic monoclonal antibodies (mAbs) or therapeutic immunoglobulins (IGs) aggregated from donors. When preparing and using this invention, those skilled in the biomedical field can use MATES materials and methods as a guide for predictable outcomes.

[0452] This agent may be referred to as a MATE agent or simply MATE. MATE agents are described, for example, in International Patent Publication WO 2021 / 102052, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the agent comprises an antibody portion, a cell receptor binding portion, and a connector portion linking the antibody portion and the cell receptor binding portion.

[0453] In one embodiment, the IgG4 binding portion is from Thermo Scientific. ® CaptureSelect ®Biotin-anti-IgG4 (human) conjugate (catalog number 7102902500 or catalog number 7102902100). CaptureSelect ® The biotin-anti-IgG4 (human) conjugate contains a 12 kDa recombinant single-domain antibody fragment (VHH affinity ligand) that specifically binds to the Fc region of the human IgG4 subclass. The inventors bind the 12 kDa recombinant antibody fragment via biotin conjugation. The VHH affinity ligand contains three complementarity-determining regions (CDRs) that form the antigen-binding domain. The affinity ligand is chemically conjugated to biotin via a suitable spacer, which preserves the ligand's binding reactivity when used in combination with streptavidin-based conjugates or streptavidin-pre-coated surfaces.

[0454] Exemplary procedures for conjugation and reaction kinetic analysis .

[0455] Buffer exchange Five mg of protein via Amicon ® The filter undergoes buffer exchange and concentrates the solution to the target concentration.

[0456] Adhesion Add 1.5 mg of concentrated antibody to a 1.5 mL tube, and add reagent (10 mM dimethyl sulfoxide solution). The total concentration of dimethyl sulfoxide in the reaction mixture is 3.74%. The reaction concentration is 10 mg / mL.

[0457] Incubation Bring the tube to near ambient temperature, then incubate at 25°C.

[0458] Sampling and Analysis At each of the seven reaction times (0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h), 10 μL of sample was quenched with four equivalents of propylamine and then frozen at -80 °C. After the last sample was quenched and frozen at -80 °C for a few minutes, samples were thawed individually and analyzed by whole liquid chromatography-mass spectrometry (LC-MS) to obtain MS-DAR. Purity was also determined by size exclusion chromatography-high performance liquid chromatography.

[0459] Using 50 mM HEPES, pH 7.0 as the reaction buffer, the ADC reacted violently in the first two hours, and the 3x reagent approached DAR6 within five hours. Measurements were taken by liquid chromatography-mass spectrometry.

[0460] Aggregation of varying degrees occurred when 50 mM HEPES, pH 7.0 was used as the reaction buffer and quenched with tetraequivalents of propylamine. Measurements were performed using size exclusion chromatography.

[0461] Joining process scheme .

[0462]

[0463] Multiple medications. Second medication. .

[0464] In another embodiment, the present invention provides a composition comprising an agent; and at least one additional agent comprising an antibody portion capable of binding to an antibody portion forming an antibody portion of a first compound.

[0465] In some embodiments, the first composition is a composition comprising a first pharmaceutical agent as described in this specification. In some embodiments, the second pharmaceutical agent independently comprises a second reactive group. In some embodiments, the second composition is a composition comprising multiple pharmaceutical agents as described in this specification, wherein each cell receptor binding moiety is independently a reactive group as described in this specification. In some embodiments, the second composition is an antibody composition, wherein the antibody in the composition is unmodified. In some embodiments, the second composition is an IVIG formulation. In some embodiments, the product composition is a composition comprising multiple pharmaceutical agents as described in this specification, wherein each cell receptor binding moiety is independently a cell receptor binding moiety as described in this specification.

[0466] In some embodiments, the cell receptor-binding portion of the product drug is the cell receptor-binding portion of the first drug. In some embodiments, the antibody portion of the product drug is the antibody portion of the second drug. In some embodiments, the second drug is an antibody drug, such as an antibody in a monoclonal antibody, a polyclonal antibody, an antibody in an IVIG formulation, etc. In some embodiments, the second reactive group is a functional group of an amino acid residue, such as −NH2 of Lys, −SH of Cys, etc. In some embodiments, the second reactive group is −NH2 of a Lys residue, for example, −NH2 of residues selected from: K246 and K248 of the IgG1 heavy chain and their corresponding amino acid residues, K251 and K253 of the IgG2 heavy chain and their corresponding amino acid residues, and K239 and K241 of the IgG4 heavy chain and their corresponding amino acid residues. In some embodiments, the present invention provides selective reactivity at the amino acid residues of the antibody portion.

[0467] In some embodiments, a second reactive group is optionally attached to the antibody portion via a connector. In some embodiments, the second reactive group is selectively attached to a position on the antibody portion, for example, selected from: K246 and K248 of the IgG1 heavy chain, and their corresponding amino acid residues; K251 and K253 of the IgG2 heavy chain, and their corresponding amino acid residues; and K239 and K241 of the IgG4 heavy chain, and their corresponding amino acid residues. In some embodiments, the present invention provides selective reactivity at amino acid residues on the antibody portion.

[0468] In some embodiments, the present invention provides agents, each independently comprising an antibody-binding portion, a reactive group, a cell receptor-binding portion, and optionally one or more connector portions connecting such groups / portions. In some embodiments, such agents can be used as reactive couplers (e.g., a first agent) for conjugating a portion of interest, such as a target-binding portion, a reactive group (e.g., a second reactive group), to an agent containing an antibody portion (e.g., a second agent). In some embodiments, the present invention provides agents for conjugating a portion of interest to an antibody portion in several agents or antibody agents (e.g., monoclonal antibody agents, polyclonal antibody agents, antibody agents of IVIG formulations, etc.). In some embodiments, the provided agents each comprise a cell receptor-binding portion, a reactive group, an antibody-binding portion, and optionally one or more connector portions (connectors) connecting such portions. In some embodiments, the antibody-binding portion is a leaving group portion that is released after contacting the agent (e.g., a first agent) with an antibody portion (e.g., an antibody portion of a second agent) and reacting a reactive group of the agent (e.g., a first reactive group of the first agent) with a reactive group of the antibody portion (e.g., a second reactive group of the second agent, such as −NH2 of a Lys residue of an antibody protein). In some embodiments, the provided technology can provide the conjugated product agent with improved conjugation efficiency, high selectivity, and / or fewer steps (sometimes, a single step). In some embodiments, the provided agent, such as the first agent, is a composition of the formula AGN301 or a salt thereof:

[0469] LG−RG−LRM−TBT,

[0470] [AGN301] or its salt, wherein:

[0471] LG is a group that contains the antibody-binding moiety;

[0472] RG is a reactive group;

[0473] LRM is a connector; and

[0474] TBT is the cell receptor binding site.

[0475] In some embodiments, the present invention provides a composition comprising a plurality of agents, wherein each agent independently comprises:

[0476] Antibody portion,

[0477] Cell receptor binding site, and

[0478] Optionally, a connector portion connects the antibody-binding portion and the cell receptor-binding portion.

[0479] In some embodiments, the product agent is a MATE agent. In some embodiments, the antibody agent portion includes an IgGFc region. In some embodiments, the antibody portion is linked to a cell receptor binding portion via an amino group, optionally via a linker. In some embodiments, the antibody portion is linked to a lysine residue, wherein the amino group of the side chain is optionally linked to a cell receptor binding portion (e.g., forming −NH−C(O)− as an amide group, carbamate group, etc.) via a linker.

[0480] In some embodiments, a selected position of the antibody portion is used for conjugation. In some embodiments, K246 or K248 (EU number or corresponding residue) of the antibody agent is the conjugation position. In some embodiments, the conjugation position is K246 of the heavy chain (unless otherwise stated, positions in this specification include, for example, the corresponding residue in a modified sequence (e.g., longer, shorter, rearranged, etc.). In some embodiments, the position is K248 of the heavy chain. In some embodiments, the position is K288 or K290 of the heavy chain. In some embodiments, the position is K288 of the heavy chain. In some embodiments, the position is K290 of the heavy chain. In some embodiments, the position is K317. In some embodiments, the antibody portion is a portion of an IgG1 antibody or a fragment thereof. In some embodiments, the antibody portion is a portion of an IgG2 antibody or a fragment thereof. In some embodiments, the antibody portion is a portion of an IgG4 antibody or a fragment thereof. In some embodiments, the composition comprises multiple MATE agents, wherein the antibody portions of the multiple MATE agents are independently antibody portions of IgG1, IgG2, or IgG4 antibodies or fragments thereof.

[0481] In some embodiments, the antibody heavy chain is selectively conjugated / labeled relative to the light chain.

[0482] This specification can provide controlled cell receptor binding moieties, such as cell receptor binding moieties / antibody moieties ratios, for example, for a cell receptor binding moieties that are peptide cell receptor binding moieties, the peptide cell receptor binding moieties / antibody ratio (PAR). In some embodiments, the ratio is from about 0.1 to 10, 0.5 to 6, etc., for example from about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 to about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5. 5, 6, 7, 8, 9, 10, approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6, 7, 8, 9, 10, etc. In some embodiments, the ratio is the ratio of the portion of interest conjugated to the antibody portion to the antibody portion conjugated to the portion of interest, for example, when the ratio is a pharmaceutical ratio. In some embodiments, the ratio is the ratio of the portion of interest conjugated to the antibody portion to all antibodies in the composition, for example, when the ratio is a composition ratio. In some embodiments, the ratio is approximately 0.1 to 6. In some embodiments, the ratio is approximately 0.5 to 2.5. In some embodiments, the ratio is about 0.5 to 2. In some embodiments, the ratio is about 1 to 2. In some embodiments, the ratio is about 1.5 to 2. In some embodiments, the ratio of IgG1, IgG2, and / or IgG4 antibodies or fragments thereof is about 1.5 to 2. In some embodiments, for the composition, such as the target binding portion conjugated to IVIG, the ratio is about 1.5 to 2.5. In some embodiments, the ratio is about 0.1. In some embodiments, the ratio is about 0.2. In some embodiments, the ratio is about 0.3. In some embodiments, the ratio is about 0.4. In some embodiments, the ratio is about 0.5. In some embodiments, the ratio is about 0.6. In some embodiments, the ratio is about 0.7. In some embodiments, the ratio is about 0.8. In some embodiments, the ratio is about 0.9. In some embodiments, the ratio is about 1. In some embodiments, the ratio is about 1.1. In some embodiments, the ratio is about 1.2. In some embodiments, the ratio is about 1.3. In some embodiments, the ratio is about 1.4. In some embodiments, the ratio is about 1.5. In some embodiments, the ratio is about 1.6. In some embodiments, the ratio is about 1.7. In some embodiments, the ratio is about 1.8. In some embodiments, the ratio is about 1.9. In some embodiments, the ratio is about 2.In some embodiments, the ratio is about 2.1. In some embodiments, the ratio is about 2.2. In some embodiments, the ratio is about 2.3. In some embodiments, the ratio is about 2.4. In some embodiments, the ratio is about 2.5. In some embodiments, for compositions in which the antibody portion of multiple agents is the antibody portion of an IVIG formulation, the ratio is about 1.8. In some embodiments, the ratio of the target binding portion to the antibody portion is about 1.5-2, wherein the antibody portion of multiple agents is the antibody portion of IgG1. In some embodiments, the ratio of the target binding portion to the antibody portion is about 1.5-2, wherein the antibody portion of multiple agents is the antibody portion of IgG2. In some embodiments, the ratio of the target binding portion to the antibody portion is about 1.5-2.0, wherein the antibody portion of multiple agents is the antibody portion of IgG4. In some embodiments, the ratio is about 1.9 to 2.0.

[0483] In some embodiments, in the provided reagent (e.g., a reagent of formula AGN101 or AGN102 or a salt thereof), substantially all conjugation sites of the antibody portion have the same modification, e.g., all share the same portion of interest optionally linked by the same linker portion. In some embodiments, no conjugation sites have different modifications, e.g., different portions of interest and / or no portions of interest and / or different linker portions.

[0484] In some embodiments, approximately 10% to 100% of all or substantially all of the portion of interest (e.g., the target-binding portion) conjugated to an antibody portion of a particular type of antibody (e.g., IgG1) or a fragment thereof is conjugated to one or more specific sites, typically one or two specific sites, such as K246 and K248 of the IgG1 heavy chain and their corresponding amino acid residues. In some embodiments, approximately 10% to 100% of all or substantially all of the portion of interest (e.g., the target-binding portion) conjugated to an antibody portion of an IgG2 antibody or a fragment thereof is located at K251 and K253 of the IgG2 heavy chain and their corresponding amino acid residues. In some embodiments, approximately 10% to 100% of all or substantially all of the portion of interest (e.g., the target-binding portion) conjugated to an antibody portion of an IgG2 antibody or a fragment thereof is located at K239 and K241 of the IgG4 heavy chain and their corresponding amino acid residues. In some embodiments, such as for multiple pharmaceutical agents, for compositions, etc., about 10% to 100% of all or substantially all of the portion of interest is conjugated to the antibody portion of an IgG1, IgG2, and / or IgG4 antibody or a fragment thereof, for example, for conjugated products with an IgG1 antibody or a fragment thereof (the antibody portion is an IgG1 antibody or a fragment thereof), with an IgG2 antibody or a fragment thereof (the antibody portion is an IgG2 antibody or a fragment thereof), with an IgG4 antibody or a fragment thereof (the antibody portion is an IgG4 antibody or a fragment thereof), or with an IVIG (selectively conjugated with IgG1, IgG2, and IgG4 when using some of the techniques provided in this specification). In some embodiments, the percentage is about 10% or more. In some embodiments, the percentage is about 20% or more. In some embodiments, the percentage is about 25% or more. In some embodiments, the percentage is about 30% or more. In some embodiments, the percentage is about 40% or more. In some embodiments, the percentage is about 50% or more. In some embodiments, the percentage is about 60% or more. In some embodiments, the percentage is about 65% or more. In some embodiments, the percentage is about 70% or more. In some embodiments, the percentage is about 75% or more. In some embodiments, the percentage is about 80% or more. In some embodiments, the percentage is about 85% or more. In some embodiments, the percentage is about 90% or more. In some embodiments, the percentage is about 95% or more. In some embodiments, the percentage is about 100%.

[0485] In some embodiments, the composition comprises multiple agents, such as MATE agents, agents of formula AGN101 or AGN102, or salts thereof, each independently comprising a cell receptor binding portion, an antibody portion, and optionally a linker portion connecting the cell receptor binding portion and the antibody portion. In some embodiments, substantially all target binding portions of the multiple agents are identical. In some embodiments, substantially all target binding portions of the multiple agents comprise a peptide portion with a common amino acid sequence. In some embodiments, substantially all target binding portions of the multiple agents are peptide portions with a common amino acid sequence. In some embodiments, substantially all conjugation sites of the antibody portions in the multiple agents have the same modification, e.g., all share the same portion of interest optionally linked by the same linker portion. In some embodiments, no conjugation sites of the multiple agents have different modifications, e.g., different portions of interest and / or no portions of interest and / or different linker portions. In some embodiments, the multiple agents do not have agents that share the same (or substantially the same) antibody portion but have different modifications (e.g., different portions of interest and / or no portions of interest and / or different linker portions). In some embodiments, agents that share the same (or substantially the same) antibody moiety but have different modifications (e.g., different moieties of interest and / or no moieties of interest and / or different linker moieties) are intermediates in the multi-step preparation of the final product agent (e.g., including steps for removing the antibody-binding moiety and steps for conjugating the cell receptor-binding moiety).

[0486] In some embodiments, the antibody portion is a portion of an IgG1 antibody or a fragment thereof. In some embodiments, the antibody portion is a portion of an IgG2 antibody or a fragment thereof. In some embodiments, the antibody portion is a portion of an IgG3 antibody or a fragment thereof. In some embodiments, the antibody portion is a portion of an IgG4 antibody or a fragment thereof. In some embodiments, approximately 1% to 100% of all portions of interest are located at a common location. In some embodiments, the cell receptor binding portion is a cell receptor binding portion as described in this specification. In some embodiments, multiple agents each independently have the formula AGN101 or AGN102 or a salt thereof.

[0487] In some embodiments, the antibody portions of multiple agents contain a common amino acid sequence. In some embodiments, the antibody portions of multiple agents contain a common amino acid sequence in the Fc region. In some embodiments, the antibody portions of multiple agents contain a common Fc region. In some embodiments, the antibody portions of multiple agents can specifically bind to a common antigen. In some embodiments, the antibody portion is a monoclonal antibody portion. In some embodiments, the antibody portion is a polyclonal antibody portion. In some embodiments, the antibody portion binds to two or more different antigens. In some embodiments, the antibody portion binds to two or more different proteins. In some embodiments, the antibody portion is an IVIG portion.

[0488] In some embodiments, the cell receptor binding portion of one of a plurality of agents is a cell receptor binding portion. In some embodiments, each cell receptor binding portion is independently a cell receptor binding portion. In some embodiments, the composition comprises a plurality of agents, the antibody portions of the plurality of agents comprising a common amino acid sequence, and the plurality of agents sharing a common cell receptor binding portion, each of which is independently and optionally independently linked to a common amino acid residue in the common amino acid sequence via a linker; and approximately 1% to 100% of all agents comprising antibody portions containing a common amino acid sequence and a common cell receptor binding portion independently comprises a common cell receptor binding portion, which is independently and optionally linked to a common amino acid residue via a linker. In some embodiments, the composition comprises multiple agents, the antibody portions of the multiple agents comprising a common amino acid sequence, and the multiple agents sharing a common cell receptor binding portion, each cell receptor binding portion being independently linked to a common amino acid residue in the common amino acid sequence via a linker; and approximately 1% to 100% of all agents comprising antibody portions containing a common amino acid sequence and a common cell receptor binding portion independently comprises a common cell receptor binding portion, which is independently linked to a common amino acid residue in the common amino acid sequence via a linker. In some embodiments, the composition comprises multiple agents, the antibody portions of the multiple agents comprising a common amino acid sequence, and the multiple agents sharing a common cell receptor binding portion, each cell receptor binding portion being independently and optionally independently linked to a common amino acid residue in the common amino acid sequence via a common linker; and approximately 1% to 100% of all agents comprising antibody portions containing a common amino acid sequence and a common cell receptor binding portion are multiple agents. In some embodiments, the composition comprises multiple agents, wherein the antibody portions of the multiple agents contain a common amino acid sequence, and the multiple agents share a common cell receptor binding portion, each of which is independently linked to a common amino acid residue in the common amino acid sequence via a linker; and approximately 1% to 100% of all agents comprising antibody portions containing a common amino acid sequence, a common cell receptor binding portion, and a common linker are multiple agents.

[0489] In some embodiments, "at least one" or "one or more" is 1-1000, 1-500, 1-200, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In some embodiments, it is one. In some embodiments, it is two or more. In some embodiments, it is about 3. In some embodiments, it is about 4. In some embodiments, it is about 5. In some embodiments, it is about 6. In some embodiments, it is about 7. In some embodiments, it is about 8. In some embodiments, it is about 9. In some embodiments, it is about 10. In some embodiments, it is about 10 or more.

[0490] In some embodiments, the common amino acid sequence comprises 1 to 1000, 1 to 500, 1 to 400, 1 to 300, 1 to 200, 1 to 100, 1 to 50, 10 to 1000, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 20 to 1000, 20 to 500, 20 to 400, 20 to 300, 20 to 200, 20 to 100, 20 to 50, 50 to 1000, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 400, 500, 600 or more amino acid residues. In some embodiments, the length is at least 5 amino acid residues. In some embodiments, the length is at least 10 amino acid residues. In some embodiments, the length is at least 50 amino acid residues. In some embodiments, the length is at least 100 amino acid residues. In some embodiments, the length is at least 150 amino acid residues. In some embodiments, the length is at least 200 amino acid residues. In some embodiments, the length is at least 300 amino acid residues. In some embodiments, the length is at least 400 amino acid residues. In some embodiments, the length is at least 500 amino acid residues. In some embodiments, the length is at least 600 amino acid residues.

[0491] In some embodiments, the common amino acid sequence is at least 10% to 100%, 50% to 100%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of the antibody moiety, protein pharmaceutical moiety, etc. In some embodiments, it is 10% or higher. In some embodiments, it is 20% or higher. In some embodiments, it is 30% or higher. In some embodiments, it is 40% or higher. In some embodiments, it is 50% or higher. In some embodiments, it is 60% or higher. In some embodiments, it is 70% or higher. In some embodiments, it is 80% or higher. In some embodiments, it is 90% or higher. In some embodiments, it is 100%.

[0492] In some embodiments, one and only one amino acid residue in the common amino acid sequence is linked to a common cell receptor binding moiety, such as the common cell receptor binding moiety. In some embodiments, two and only two amino acid residues in the common amino acid sequence are linked to a common cell receptor binding moiety, such as the common cell receptor binding moiety. In some embodiments, two or more amino acid residues in the common amino acid sequence are linked to a common cell receptor binding moiety, such as the common cell receptor binding moiety. In some embodiments, each common cell receptor binding moiety (e.g., the common cell receptor binding moiety) is independently linked to amino acid residues in the common amino acid sequence.

[0493] In some embodiments, the common amino acid sequence comprises one or more amino acid residues selected from: K246 and K248 of the IgG1 heavy chain and their corresponding amino acid residues, K251 and K253 of the IgG2 heavy chain and their corresponding amino acid residues, and K239 and K241 of the IgG4 heavy chain and their corresponding amino acid residues. In some embodiments, the common amino acid sequence comprises one or more amino acid residues selected from: K246 and K248 of the IgG1 heavy chain and their corresponding amino acid residues. In some embodiments, the common amino acid sequence comprises one or more amino acid residues selected from: K251 and K253 of the IgG2 heavy chain and their corresponding amino acid residues. In some embodiments, the common amino acid sequence comprises one or more amino acid residues selected from: K239 and K241 of the IgG4 heavy chain and their corresponding amino acid residues. In some embodiments, the cell receptor binding moiety is linked to the amino acid residue (optionally via a linker portion unless explicitly stated). In some embodiments, each cell receptor binding moiety is optionally and independently linked to the amino acid residue via a linker portion.

[0494] In some embodiments, the antibody portions share a high percentage of amino acid sequence homology. In some embodiments, the amino acid sequence homology is approximately 50%–100%. In some embodiments, the amino acid sequence homology is a percentage selected from the Markush percentage group consisting of: 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

[0495] In some embodiments, the percentages used in this specification are percentages selected from the Markush percentage group consisting of: about 1% to 100%, about 10% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 100%.

[0496] In some embodiments, the antibody portions of multiple agents contain a common Fc region or a fragment thereof.

[0497] In some embodiments, the portions of interest of multiple agents are located at specific positions. In some embodiments, all portions of interest are located at amino acid residues of a common amino acid sequence. In some embodiments, all portions of interest are located at common positions of amino acid residues of a common amino acid sequence. In some embodiments, the number of common positions is 1. In some embodiments, it is 2. In some embodiments, it is 3. In some embodiments, it is 4. In some embodiments, the antibody portion comprises two heavy chains or fragments thereof, and the number of common positions is 2 (one on each chain). In some embodiments, the common positions are selected from: K246 and K248 of the IgG1 heavy chain and their corresponding amino acid residues, K251 and K253 of the IgG2 heavy chain and their corresponding amino acid residues, and K239 and K241 of the IgG4 heavy chain and their corresponding amino acid residues.

[0498] In some embodiments, multiple agents independently share a common cell receptor binding portion at at least one location. In some embodiments, multiple agents independently share a common cell receptor binding portion and a connector at at least one location. In some embodiments, the portion of interest at two or more or all locations includes a common cell receptor binding portion. In some embodiments, the portion of interest is identical.

[0499] In some embodiments, the agents share a common modification at at least one common amino acid residue. In some embodiments, multiple agents share a common modification at each site where they are attached to the cell receptor binding site and optionally the linker. In some embodiments, multiple agents have the same −L at each site where they are attached to the linker site. PM −TBT.

[0500] In some embodiments, the position is selected from K246, K248, K288, K290, K317 of the antibody agent and their corresponding positions. In some embodiments, the position is selected from K246 and K248 and their corresponding positions. In some embodiments, the position is selected from K288 and K290 and their corresponding positions. In some embodiments, the position is K246 or its corresponding position. In some embodiments, the position is K248 or its corresponding position. In some embodiments, the position is K288 or its corresponding position. In some embodiments, the position is K290 or its corresponding position. In some embodiments, the position is K317 or its corresponding position. In some embodiments, the position is K185 of the light chain or its corresponding position. In some embodiments, the position is K187 of the light chain or its corresponding position. In some embodiments, the position is K133 of the heavy chain or its corresponding position. In some embodiments, the position is K246 or K248 of the heavy chain or its corresponding position. In some embodiments, the position is K414 of the heavy chain or its corresponding position. In some embodiments, the common sequence is a sequence of about or at least about 10 to 100, 20 to 50, such as about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, and contains one or more such residues or their corresponding residues. In some embodiments, the common sequence is a sequence of about or at least about 10 to 100, 20 to 50, such as about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, and contains one, two, or more residues selected from: K246 and K248 of the IgG1 heavy chain and their corresponding amino acid residues, K251 and K253 of the IgG2 heavy chain and their corresponding amino acid residues, and K239 and K241 of the IgG4 heavy chain and their corresponding amino acid residues.

[0501] In some embodiments, about 1% to 100% of all agents comprising an antibody portion and a cell receptor binding portion are multiple agents. In some embodiments, about 1% to 100% of all agents comprising an antibody portion and a cell receptor binding portion containing a common amino acid sequence are multiple agents. In some embodiments, about 1% to 100% of all agents comprising an antibody portion and a cell receptor binding portion containing a common amino acid sequence or capable of binding to a common antigen are multiple agents. In some embodiments, about 1% to 100% of all agents comprising an antibody portion are multiple agents. In some embodiments, about 1% to 100% of all agents comprising an antibody portion containing a common amino acid sequence are multiple agents. In some embodiments, about 1% to 100% of all agents comprising a protein agent portion containing a common amino acid sequence are multiple agents. In some embodiments, the range of all agents comprising an antibody agent portion containing a common amino acid sequence or capable of binding to a common antigen is selected from the Markush range, wherein multiple agents account for about 1% to 100%, about 5% to 100%, 10% to 100%, about 20% to 100%, about 25% to 100%, about 30% to 100%, and about 40% to 100%, about 50% to 100%. In some embodiments, the range of all agents comprising an antibody agent portion containing a common amino acid sequence or capable of binding to a common antigen is selected from the Markush range, wherein multiple agents account for about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 100%. In some embodiments, the range of all agents comprising an antibody agent portion containing a common amino acid sequence or capable of binding to a common antigen is selected from the Markush range group, wherein multiple agents account for at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99%.

[0502] In some embodiments, each of the plurality of pharmaceutical agents does not have −S−Cy−, wherein −Cy− is an optionally substituted 5-membered monocyclic ring, does not have −S−S− which is not formed of cysteine ​​residues, and does not have −SH or its salt form which is not a cysteine ​​residue. In some embodiments, each of the plurality of pharmaceutical agents does not have −S−CH2−CH2−. In some embodiments, each of the plurality of pharmaceutical agents does not have a portion that can specifically bind to an antibody pharmaceutical agent. In some embodiments, the composition is substantially free of a portion that can specifically bind to an antibody pharmaceutical agent.

[0503] In some embodiments, the provided agents, compounds, etc., such as those of formula AGN301, AGN101, AGN102, etc., and their salts, have high purity. In some embodiments, the percentage is about 5%-100%. In some embodiments, the percentage is about 10%-100%. In some embodiments, the percentage is about 20%-100%. In some embodiments, the percentage is about 25%-100%. In some embodiments, the percentage is about 30%-100%. In some embodiments, the percentage is about 40%-100%. In some embodiments, the percentage is about 50%-100%. In some embodiments, it is about 5%. In some embodiments, it is about 10%. In some embodiments, it is about 20%. In some embodiments, it is about 25%. In some embodiments, it is about 30%. In some embodiments, it is about 40%. In some embodiments, it is about 50%. In some embodiments, it is about 60%. In some embodiments, it is about 70%. In some embodiments, it is about 80%. In some embodiments, it is about 90%. In some embodiments, it is about 91%. In some embodiments, it is about 50%. In some embodiments, it is about 92%. In some embodiments, it is about 93%. In some embodiments, it is about 94%. In some embodiments, it is about 95%. In some embodiments, it is about 96%. In some embodiments, it is about 97%. In some embodiments, it is about 98%. In some embodiments, it is about 99%. In some embodiments, it is about 100%. In some embodiments, it is at least about 5%. In some embodiments, it is at least about 10%. In some embodiments, it is at least about 20%. In some embodiments, it is at least about 25%. In some embodiments, it is at least about 30%. In some embodiments, it is at least about 40%. In some embodiments, it is at least about 50%. In some embodiments, it is at least about 60%. In some embodiments, it is at least about 70%. In some embodiments, it is at least about 80%. In some embodiments, it is at least about 90%. In some embodiments, it is at least about 90%. In some embodiments, it is at least about 91%. In some embodiments, it is at least about 50%. In some embodiments, it is at least about 92%. In some embodiments, it is at least about 93%. In some embodiments, it is at least about 94%. In some embodiments, it is at least about 95%. In some embodiments, it is at least about 96%. In some embodiments, it is at least about 97%. In some embodiments, it is at least about 98%. In some embodiments, it is at least about 99%.

[0504] In some embodiments, the present invention provides a product pharmaceutical composition comprising a product agent (e.g., an agent of formula AGN101 or AGN102) or a salt thereof. In some embodiments, the product pharmaceutical composition (e.g., a pharmaceutical composition formed by certain methods) comprises a product agent containing an antibody portion and a cell receptor binding portion, and optionally a linker (e.g., an agent of formula AGN101 or AGN102 or a salt thereof), and a released antibody binding portion, for example containing R... LG −(L LG1 ) 0-1 −(L LG2 ) 0-1 −(L LG3 ) 0-1 −(L LG4 ) 0-1 Compounds containing an antibody-binding moiety, such as those having R- LG −(L LG1 ) 0-1 −(L LG2 ) 0-1 −(L LG3 ) 0-1 −(L LG4 ) 0-1 Compounds with an -H structure or salts thereof, and reaction couplers, such as a composition of the formula AGN301 or a salt thereof. In some embodiments, the released antibody-binding portion may bind to the antibody portion in the target agent and / or the formed product agent. According to this specification, several techniques can be used to separate the released antibody-binding portion from the antibody portion, for example, in some embodiments, contacting the composition with a composition containing glycine at a specific pH. In some embodiments, each of a plurality of agents is independently the product agent.

[0505] reactive groups

[0506] In some embodiments, the provided agent or compound, such as those that can be used as a reaction partner (e.g., the first agent), contains a reactive group (e.g., RG). In some embodiments, the reactive group (e.g., RG) is located between the antibody-binding portion (e.g., ABT) and the portion of interest (e.g., MOI), and optionally and independently connected to both the antibody-binding portion and the portion of interest via a linker. In some embodiments, RG is a reactive group as described in this specification.

[0507] In some embodiments, when used in a pharmaceutical agent that does not contain an antibody-binding moiety, the reactive group reacts slowly and provides low levels, in some embodiments, substantially no conjugation of the moiety of interest to the target agent. As illustrated in this specification, the combination of the reactive group and the antibody-binding moiety in the same pharmaceutical agent (e.g., in a compound of formula AGN301 or a salt thereof) can promote the reaction between the reactive group and the target agent, enhance reaction efficiency, reduce side reactions, and / or improve reaction selectivity (e.g., with respect to the target site in which the moiety of interest conjugates to the target agent).

[0508] The reactive groups in the pharmaceutical agent can react with several types of groups in the target pharmaceutical agent. In some embodiments, the reactive groups in the pharmaceutical agent selectively react with amino groups of the target pharmaceutical agent, such as −NH2 groups on the side chains of lysine residues of proteins. In some embodiments, when used in a pharmaceutical agent (e.g., those of formula AGN301 or its salts), the reactive groups selectively react with specific sites of the target pharmaceutical agent, such as one or more of K246, K248, K288, K290, K317, etc. of IgG1, K251, K253, etc. of IgG2, K239, K241, etc. of IgG4, as illustrated in the examples of this specification. In some embodiments, the site is K246 or K248 of the antibody heavy chain. In some embodiments, the site is K246 and / or K248 of the antibody heavy chain. In some embodiments, the site is K246 of the antibody heavy chain. In some embodiments, the site is K248 of the antibody heavy chain. In some embodiments, the site is K288 or K290 of the antibody heavy chain. In some embodiments, the site is K288 of the antibody heavy chain. In some embodiments, the site is K290 of the antibody heavy chain. In some embodiments, the site is K317. In some embodiments, the site is K414 of the antibody heavy chain. In some embodiments, the site is K185 of the antibody light chain. In some embodiments, the site is K187 of the antibody light chain. In some embodiments, the site is K251 and / or K253 of the IgG2 heavy chain. In some embodiments, the site is K251 of the IgG2 heavy chain. In some embodiments, the site is K253 of the IgG2 heavy chain. In some embodiments, the site is K239 and / or K241 of the IgG4 heavy chain. In some embodiments, the site is K239 of the IgG4 heavy chain. In some embodiments, the site is K241 of the IgG4 heavy chain. In some embodiments, conjugation selectively occurs at one or more heavy chain sites instead of light chain sites. In some embodiments, for techniques without an antibody-binding portion, more conjugations occur at light chain sites than at heavy chain sites.

[0509] In some embodiments, the reactive group, such as RG, is or comprises an ester group. In some embodiments, the reactive group, such as RG, is or comprises an electrophilic group, such as a Michael receptor.

[0510] In some embodiments, the reactive group (e.g., RG) is or contains −L RG1 -L RG2 -, where L RG1 and L RG2 Each of them is independently L. In some embodiments, the reactive group (e.g., RG) is or contains −L. LG4 -L RG1 -L RG2 - where each variable is as described in this specification. In some embodiments, the reactive group (e.g., RG) is or contains -L LG3 -L LG4 -L RG1 -L RG2 - where each variable is as described in this specification. In some embodiments, the reactive group (e.g., RG) is or contains -L LG2 -L LG3 -L LG4 -L RG1 -L RG2 - where each variable is as described in this specification. In some embodiments, the reactive group (e.g., RG) is or contains -L LG4 -L RG2 - where each variable is as described in this specification. In some embodiments, the reactive group (e.g., RG) is or contains -L LG3 -L LG4 -L RG2 - where each variable is as described in this specification. In some embodiments, the reactive group (e.g., RG) is or contains -L LG2 -L LG3 -L LG4 -L RG2 - where each variable is as described in this specification.

[0511] In some embodiments, L LG4 It is −O−. In some embodiments, L LG4 It is −N(R)−. In some embodiments, L LG4 It is −NH−.

[0512] In some embodiments, L LG3 It may contain, or optionally substituted, aryl rings. In some embodiments, L LG3 It may contain a phenyl ring. In some embodiments, the aryl or phenyl ring is substituted. In some embodiments, the substituent is an electron-withdrawing group as described in this specification, such as −NO2, −F, etc.

[0513] In some embodiments, L RG1It is a covalent bond. In some embodiments, L RG1 It is not a covalent bond. In some embodiments, L RG1 It is −S(O)2−.

[0514] In some embodiments, L RG2 It is −C(O)−. In some embodiments, the reactive group is or contains −L LG4 −C(O)−, where each variable is as described in this specification. In some embodiments, the reactive group is or contains −L LG3 -L LG4 −C(O)−, where each variable is as described in this specification. In some embodiments, the reactive group is or contains −L LG2 -L LG3 -L LG4 −C(O)−, where each variable is as described in this specification.

[0515] In some embodiments, L RG2 It is −L RG3 −C(=CR RG1 R RG2 )−CR RG3 R RG4 -, where R RG1 R RG2 R RG3 and R RG4 Each of them is independently −L−R', and L RG3 It is −C(O)−, −C(O)O−, −C(O)N(R')−, −S(O)−, −S(O)2−, −P(O)(OR')−, −P(O)(SR')−, or −P(O)(N(R')2)−. In some embodiments, R RG1 R RG2 R RG3 and R RG4 Each of these is independently R'. In some embodiments, R RG1 R RG2 R RG3 and R RG4 One or more of them are independently −H. In some embodiments, L RG3 It is −C(O)−. In some embodiments, L RG3 It is −C(O)O−. In some embodiments, L RG3 -O−, -N(R')−, etc., and L PM Bonding.

[0516] In some embodiments, R RG1 It is −H. In some embodiments, R RG3 It is −H.

[0517] In some embodiments, L RG2 It is an optional substitution of −L RG3 -C(=CHR) RG2 )−CHR RG4 - where each variable is as described in this specification.

[0518] In some embodiments, R RG2 and R RG4 Together with its intermediate atom, it forms an optionally substituted ring as described in this specification. In some embodiments, the formed ring is an optionally substituted 3-10 membered monocyclic or bicyclic ring having 0-5 heteroatoms. In some embodiments, the formed ring is an optionally substituted 3-10 membered alicyclic ring. In some embodiments, the formed ring is selected from the Markush group and consists of optionally substituted alicyclic rings, which consist of 3-8 membered alicyclic rings, 5-8 membered alicyclic rings, 5 membered alicyclic rings, 6 membered alicyclic rings, and 7 membered alicyclic rings. In some embodiments, the formed ring is substituted. In some embodiments, the formed ring is not substituted. In some embodiments, except for C(=CHR) RG2 ) or C (=CR) RG1 R RG2 Apart from the double bonds in the ring, the ring formed does not have any other unsaturation.

[0519] In some embodiments, −C(=CHR) RG2 )−CHR RG4 or −C(=CR) RG1 R RG2 )−CR RG3 R RG4 It is an optional replacement Or In some embodiments, −[C(=CHR) RG2 )−CHR RG4 ]−L RG3 -or-[C(=CR)] RG1 R RG2 )−CR RG3 R RG4 ]−L RG3 - It is an optional replacement. Or In some embodiments, −L RG1 -[C(=CHR) RG2 )−CHR RG4 ]−L RG3 -or -L RG1 -[C(=CR) RG1 R RG2 )−CR RG3 R RG4 ]−LRG3 - It is an optional replacement. In some embodiments, −L RG1 -[C(=CHR) RG2 )−CHR RG4 ]−L RG3 -or -L RG1 -[C(=CR) RG1 R RG2 )−CR RG3 R RG4 ]−L RG3 - is an optional substitution.

[0520] In some embodiments, −L LG4 -L RG2 - is -O-C(O)- or -S-C(O)-. In some embodiments, -L LG4 -L RG1 -L RG2 -is -S-C(O)-.

[0521] In some embodiments, −L LG4 -L RG2 - is -N(−)−C(O)−, where N is a ring atom of an optionally substituted heteroaryl ring. In some embodiments, -L LG4 -L RG2 - is -N(−)−C(O)−, where N is an L of a heteroaryl ring that is or contains optional substituted rings. LG4 Ring atoms. In some embodiments, −L LG4 -L RG2 - is -N(−)−C(O)−O−, where N is an L of a heteroaryl ring that is or contains optionally substituted heteroaryl rings. LG4 Ring atoms.

[0522] In some embodiments, L RG2 It is optionally substituted −CH2−C(O)−, wherein −CH2− is bonded to an electron-withdrawing group comprising or connected to the antibody-binding moiety. In some embodiments, L RG2 It is an optionally substituted -CH2− group bonded to an electron-withdrawing group that is contained in or linked to the antibody-binding moiety. In some embodiments, L RG1 It is an electron-withdrawing group. In some embodiments, L RG1 Choose the Markush group consisting of the following terms: −C(O)−, −S(O)−, −S(O)2−, −P(O(OR)−, −P(O(SR)−, −P(O(N(R)2)−, −OP(O(OR)−, −OP(O(SR)−, and −OP(O(N(R)2)−).

[0523] In some embodiments, L RG2It is optionally substituted −CH2−C(O)−, wherein −CH2− is bonded to a leaving group comprising or connected to the antibody-binding moiety. In some embodiments, L RG2 It is an optionally substituted −CH2− bonded to a leaving group that is contained in or linked to the antibody-binding moiety. In some embodiments, L RG1 Choose the Markush group consisting of the following terms: −O−C(O)−, −OS(O)2−, −OP(O(OR)−, −OP(O(SR)−, and −OP(O(N(R)2)−.

[0524] In some embodiments, the reactive group reacts with the amino group of the target drug. In some embodiments, the amino group is −NH2 of the side chain of a lysine residue.

[0525] In some embodiments, the target agent is a protein agent. In some embodiments, the target agent is an antibody agent. In some embodiments, the reactive group reacts with an amino acid residue of the protein or antibody agent. In some embodiments, the amino acid residue is a lysine residue. In some embodiments, the reactive group reacts with −NH2 on the side chain of the lysine residue. In some embodiments, the reactive group is or contains −C(O)−O−, which reacts with −NH2 (e.g., on the side chain of the lysine residue) and forms an amide group −C(O)−O− with −NH2.

[0526] In some embodiments, a reactive group, such as a first reactive group, a second reactive group, etc., is located at the terminal position. In some embodiments, a pharmaceutical agent (such as a first pharmaceutical agent) includes a first reactive group optionally connected to a target-binding portion via a connector portion and does not have an antibody-binding portion.

[0527] In some embodiments, the present invention provides a method for preparing a composition comprising a plurality of agents, wherein each agent independently comprises:

[0528] Antibody portion,

[0529] Cell receptor binding site, and

[0530] Optionally, a connector portion connects the antibody portion and the cell receptor binding portion;

[0531] The method includes:

[0532] Multiple agents are contacted with multiple antibody agents, each of which independently contains a reactive group.

[0533] In some embodiments, the agent containing a reactive group comprises an antibody-binding portion, a cell receptor-binding portion, and optionally a linker. In some embodiments, the agents containing reactive groups share the same cell receptor-binding portion. In some embodiments, the agents containing reactive groups share the same structure. In some embodiments, the antibody molecule has a structure, properties, and / or activity to provide an antibody portion in the agent described herein. In some embodiments, multiple antibody molecules comprise two or more IgG subclasses. In some embodiments, multiple antibody molecules comprise IgG1. In some embodiments, multiple antibody molecules comprise IgG2. In some embodiments, multiple antibody molecules comprise IgG4. In some embodiments, multiple antibody molecules comprise IgG1 and IgG2. In some embodiments, multiple antibody molecules comprise IgG1, IgG2, and IgG4. In some embodiments, multiple antibody molecules comprise IgG1, IgG2, IgG3, and IgG4. In some embodiments, multiple antibody molecules are IVIG antibody molecules.

[0534] In some embodiments, the provided agent contains reactive groups, such as In some embodiments, −C(O)− is optionally linked via a linker to the cell receptor-binding portion or the portion containing −(Xaa)y−, and the other end is linked to the antibody-binding portion. In some embodiments, It reacts with the amino group of another portion (e.g., the antibody portion) to form an amide group and release the portion that is or contains the antibody-binding portion. In some embodiments, the amino group is −NH2 of a lysine side chain. In some embodiments, −C(O)− is optionally linked to the cell receptor-binding portion or the portion containing −(Xaa)y− via a linker, and the other end is linked to R' or an optional substituent. In some embodiments, the provided agent contains an optionally substituted... Such reactive groups can be used to conjugate with detection, diagnostic, or therapeutic agents. Those skilled in the art of biomedicine will know that, according to this specification, various pharmaceutical agents and many techniques (e.g., click chemistry, reactions based on functional groups such as amino (e.g., amide formation), hydroxyl, carboxyl, etc.) can be used for conjugation.

[0535] In some embodiments, the antibody-binding portion binds to the Fc region of the antibody. In some embodiments, the reaction occurs at residues in the Fc region. In some embodiments, the target-binding portion optionally conjugates to residues in the Fc region via a linker portion. In some embodiments, the residues are Lys residues. In some embodiments, the antibody is or contains IgG1. In some embodiments, the antibody is or contains IgG2. In some embodiments, the antibody is or contains IgG4. In some embodiments, the antibody composition for the method contains IgG1 and IgG2. In some embodiments, the antibody composition for the method contains IgG1, IgG2, and IgG4. In some embodiments, the antibody composition for the method contains IgG1, IgG2, IgG3, and IgG4.

[0536] In some embodiments, the product is or contains IgG1. In some embodiments, the product is or contains IgG2. In some embodiments, the product is or contains IgG4. In some embodiments, the product composition contains IgG1 and IgG2. In some embodiments, the product composition contains IgG1, IgG2, and IgG4. In some embodiments, the product composition contains IgG1, IgG2, IgG3, and IgG4.

[0537] In some embodiments, the provided agent comprising an antibody portion provides, for example, one or substantially all of the antibody immune activities for recruiting one or more types of immune cells and / or provides short-term and long-term immune activities. In some embodiments, the provided agent comprising an antibody portion does not significantly reduce one or substantially all of the associated antibody immune activities. In some embodiments, the provided agent comprising an antibody portion improves one or substantially all of the associated antibody immune activities (e.g., compared to the antibody portion itself). In some embodiments, the provided agent provides comparable or better stability (e.g., residence time in the blood) compared to the antibody portion itself. In some embodiments, the antibody portion in the provided agent can bind to the FcRy of immune cells (e.g., several FcRy of immune effector cells for the desired immune activity; generally at comparable or better levels). In some embodiments, the antibody portion in the provided agent has comparable Fab / antigen binding capacity. In some embodiments, the antibody portion in the provided agent has comparable Fab / antigen binding capacity. In some embodiments, the antibody portion in the provided agent provides FcRn binding. In some embodiments, the antibody portion in the provided agent provides FcRn binding, for example, for antibody cycling and / or prolonging half-life. In some embodiments, the provided technology can be used to modify blood-derived IgG products because the provided technology is suitable for and can be used with all IgG subclasses.

[0538] In some embodiments, the provided method includes one of the following steps. It reacts with the amino group of the lysine side chain to form an amide bond with the antibody molecule and releases it in the form of its salt.

[0539] Methods for evaluating the chemical structure and function of pharmaceutical agents .

[0540] protein Sequencing. In one protein sequencing method, eight digests were prepared using five different enzymes (pepsin, Lys C, trypsin, chymotrypsin, and Asp N). The digests were subjected to disulfide reduction, cysteine ​​blocking, and then enzymatic digestion. The digests were analyzed by LC-MS / MS using a Thermo-Fisher Orbitrap fusion™ mass spectrometer. Peptides were characterized from the LC-MS / MS data using de novo peptide sequencing, and the peptide groups were then assembled into antibody sequences.

[0541] Affinity chromatography

[0542] Affinity capture of ABT310 using MabSelect SuRe™ resin Purify at room temperature. Select a column based on the titer and recommended loading density (approximately 20 mg / ml resin). Equilibrate five column volumes with 25 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, and pH 7.5 buffer. Load the sample.

[0543] EQ washing Wash three to five column volumes with a buffer solution of 25 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, and pH 7.5.

[0544] Triton Washing Wash five to ten column volumes with 25 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, pH 7.5, 0.05% Triton 100, and 0.05% Triton 114.

[0545] EQ washing Wash five to ten column volumes with a buffer of 25 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, pH 7.5. Elute with 0.1 M histidine-acetate elution buffer. Neutralize immediately with 1 M arginine, 400 mM succinate, pH 9.0.

[0546] Affinity capture eluent test Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for both non-reducing and reducing methods, and size exclusion chromatography-high performance liquid chromatography.

[0547] ABT502 and ABT503 were treated with Amsphere™ A3 resin. Perform affinity capture. Purify at room temperature. Select a column based on the titer and recommended loading density (5 to 10 mg / ml resin). Equilibrate five column volumes with a buffer of 25 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, pH 7.5. Load the sample.

[0548] EQ washing Wash two to five column volumes with a buffer of 25 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, pH 7.5. Elute with elution buffer of 50 mM sodium citrate, 150 mM sodium chloride, pH 3.0. Neutralize immediately with 1 M Tris-HCl, pH 9.0.

[0549] Affinity capture eluent test Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for both non-reducing and reducing methods, and size exclusion chromatography-high performance liquid chromatography.

[0550] Results: Purification of VHH-Cys (ABT502 and ABT503) and VHH-Fc fusion (ABT310) Collect the mixed culture of cells on day 7 or when viability is <60%. The cell culture medium was clarified by centrifugation at 10,000 g for 0.5 h and aseptically filtered. ABT310 was purified by affinity capture chromatography using MabSelect SuRe™ resin (Cytiva), and ABT502 and ABT503 were purified by affinity capture chromatography using Amsphere A3 resin (JSR Life Sciences). The MabSelect SuRe™ and Amsphere A3 columns were thoroughly washed (25 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 5 mM EDTA) and eluted with 100 mM 50 histidine acetate and 50 mM sodium citrate, pH 3.0, 150 mM sodium chloride, respectively. The MabSelect SuRe™ eluent was neutralized in 1 M arginine, 400 mM succinate, pH 9.0, and Amsphere A3 resin, 1 M Tris-HCl, pH 9.0.

[0551] Size exclusion chromatography

[0552] Purify at room temperature. Select a column based on protein mass and molecular weight (Superdex 200 pg for ABT310, Superdex 75 pg for ABT502 and ABT503). Equilibrate one column volume with a buffer of 20 mM histidine acetate, 150 mM sodium chloride, pH 5.5. Load the sample. Elute one column volume with a buffer of 20 mM histidine acetate, 150 mM sodium chloride, pH 5.5. Collect the target protein with the desired purity.

[0553] Size exclusion chromatography eluent test Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for both non-reducing and reducing methods, and size exclusion chromatography-high performance liquid chromatography.

[0554] Results: Purification of VHH-Cys (ABT502 and ABT503) and VHH-Fc fusion (ABT310) ABT310 and ABT502 or ABT503 samples were applied to Superdex 200 pg (Cytiva) or Superdex 75 pg (Cytiva), respectively, and eluted in 20 mM histidine acetate, pH 5.5, 150 mM sodium chloride. The fractions were then separated and pooled for use on a cation exchange column (Poros). ® XS (Thermo Fisher) was pre-equilibrated with 20 mM sodium acetate, pH 5.5 (ABT310) or 20 mM sodium acetate, pH 5.0 (ABT502 or ABT503). The column was thoroughly washed and eluted with fifty column gradients to 100% 20 mM sodium acetate, pH 5.0 / 5.5, 1 M sodium chloride. Fractions were resolved and analyzed by size exclusion chromatography-high performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (ND-PAGE), endotoxin level assays, and liquid chromatography-mass spectrometry (LC-MS) to determine molecular weight.

[0555] Cation exchange chromatography

[0556] Purification at room temperature Using Poros ® XS column. Select the column based on protein mass and the recommended loading density (10 to 20 mg / ml resin). For ABT310, the pH for cation exchange chromatography is 5.5. For ABT502 and ABT503, the pH for cation exchange chromatography is 5.0. Dilute the sample to below 5 mS / cm with 20 mM sodium acetate, pH 5.5 / 5.0. Equilibrate five column volumes with 20 mM sodium acetate, pH 5.5 / 5.0 buffer. Load the sample.

[0557] EQ washingWash 1 to 5 column volumes with 20 mM sodium acetate, pH 5.5 / 5.0 buffer. Elute with a gradient of 0% to 100% for 50 column volumes of 20 mM sodium acetate, 1 M sodium chloride, pH 5.5 / 5.0 (the gradient can be adjusted based on the pilot run). Strip 1 to 3 column volumes with 20 mM sodium acetate, 1 M sodium chloride, pH 5.5 / 5.0. Equilibrate 1 column volume with 20 mM sodium acetate, pH 5.5 / 5.0 buffer. Sterilize with 0.5 M sodium hydroxide for 30 minutes. Equilibrate 3 to 5 column volumes with 20 mM sodium acetate, pH 5.5 / 5.0 buffer.

[0558] Cation exchange chromatography eluent testing Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for both non-reducing and reducing methods, and size exclusion chromatography-high performance liquid chromatography.

[0559] SEC-HPLC

[0560] The inventors used TSKgel G3000SWxl stainless steel columns (7.8 x 300 mm, 5µm particle size) in VanquishFlex Duo. ® Size exclusion chromatography-high performance liquid chromatography (HPLC) was performed on a liquid chromatography instrument. The mobile phase consisted of 50 mM sodium phosphate and 300 mM sodium chloride at pH 6.8. 25 µg of sample was injected per run. Isocratic elution was performed at a flow rate of 1.0 mL / min for 15 minutes. Protein elution was monitored by UV absorbance at 280 nm. The peaks corresponding to aggregates, monomers, and low molecular weight substances were integrated, and the percentage of each substance was calculated.

[0561] SDS-PAGE

[0562] The inventors used pre-prepared NuPAGE™ 4-12% bis-Tris gels from Thermo Scientific for non-reducing and reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Sample loading buffer (4x lithium dodecyl sulfate) was from Invitrogen. Gel electrophoresis buffer (20x MES) was from GenScript. Non-reducing samples were treated with 30 mM iodoacetamide and heated at 95°C for five minutes prior to analysis. Reducing samples were treated with 50 mM dithiothreitol and heated at 95°C for five minutes prior to analysis. Electrophoresis was performed at a constant voltage of 180 V for forty minutes. The gels were stained with Coomassie blue for thirty minutes and then destained with water for one hour.

[0563] Molecular weight analysis by LC-MS

[0564] Liquid chromatography-mass spectrometry analysis was performed using an Agilent 6230 AdvanceBio LC / TOF system equipped with an Agilent PL1912-1502, PLRP-S 1000Å, 2.1 x 50 mm, 5 µm column (Agilent / PL1912-1502).

[0565] For non-reducing mass analysis, 200 pmol of sample was diluted with ddH₂O and LC / MS was performed. For reducing mass analysis, 100 pmol of sample was denatured and reduced using 10 mM urea (Sigma / 15568), 30 mM Tris-HCl (Invitrogen / 15568), and dithiothreitol (Sigma / D0632) in a total volume of 50 µL. Samples were incubated at 37 °C for 20 min before LC / MS. The inventors used Bioconfirm 10.0 software to analyze the mass spectrometry data and identify molecules based on molecular mass.

[0566] Endotoxin level test :

[0567] The purified protein was diluted in LAL reagent water (Charles River / W110) based on maximum effective dilution (MVD). The sample was loaded into a Charles River Laboratories EndoSafe LAL assay card (Charles River / PTS11F) and analyzed using a Charles River Laboratories Endosafe nexgen-MCS™ instrument.

[0568] Methods for evaluating the agents used for application

[0569] General technicians in the biomedical field can use the United States Pharmacopeia - National Formulary (USP) <631> Or the appearance, color and colorlessness of the pharmaceutical preparation as described in the European Pharmacopoeia Ph. Eur. 2.2.2.

[0570] General technicians in the biomedical field can use the United States Pharmacopeia - National Formulary (USP) <1061> Or the appearance and clarity of the standard analytical pharmaceutical preparation as described in the European Pharmacopoeia Ph. Eur. 2.2.2.

[0571] General technicians in the biomedical field can use the United States Pharmacopeia - National Formulary (USP) <791> Or the pH of the standard analytical pharmaceutical preparation as described in the European Pharmacopoeia Ph. Eur. 2.2.3. A generally accepted standard is ±0.5 pH of the target pH.

[0572] General technicians in the biomedical field can use the United States Pharmacopeia - National Formulary (USP) <785> Or the osmotic pressure of a standard analytical pharmaceutical product as described in the European Pharmacopoeia Ph. Eur. 2.2.35.

[0573] A person with general technical skills in the biomedical field can quantify the concentration of a drug using UV-VIS spectroscopy (e.g., at A280 (280 nm)). A commonly accepted standard is ±10% of the target concentration.

[0574] Ordinary technicians in the biomedical field can identify drugs by measuring the retention time or pI of charge variant peaks, such as by imaging capillary isoelectric focusing (iCIEF), a high-resolution technique that separates proteins into groups based on their isoelectric point (pI).

[0575] A person with general technical skills in the biomedical field can confirm the purity of a pharmaceutical agent using hydrophobic interaction chromatography-high performance liquid chromatography (HPLC). For example, a person with general technical skills in the biomedical field can confirm the DAR (Damage per Amount) of a pharmaceutical agent. A generally accepted standard is ±10% of the target concentration.

[0576] A person with general skills in the biomedical field can confirm the purity of pharmaceutical formulations using size exclusion chromatography-high performance liquid chromatography (HPLC). For example, a person with general skills in the biomedical field can determine the percentages of monomers, high molecular weight, and low molecular weight. A generally accepted standard is a result of ≥90% or ≤5%.

[0577] A person with general technical skills in the biomedical field can confirm the purity of a pharmaceutical preparation using the CE-SDS-NR method. For example, a person with general technical skills in the biomedical field can confirm the percentage of the main peak and fragment peaks. The generally accepted standard is a result ≥85%.

[0578] A person with general technical skills in the biomedical field can confirm the purity of pharmaceutical formulations using the CE-SDS-R method. For example, a person with general technical skills in the biomedical field can confirm the percentage of monomer and minor component peaks. The generally accepted standard is a result ≥85%.

[0579] A person skilled in the biomedical field can confirm the purity of a pharmaceutical formulation using imaging capillary isoelectric focusing methods. For example, a person skilled in the biomedical field can determine the percentage of charge variants.

[0580] General technicians in the biomedical field can use the ELISA method to confirm the binding potency of a drug. The generally accepted standard is a ±100% percentage compared to a reference standard.

[0581] General technicians in the biomedical field can detect process impurities (HCPs) in pharmaceutical formulations using the ELISA method. The generally accepted standard is ≤ 100 ng / mg.

[0582] General technicians in the biomedical field can use ELISA to detect process impurities (rProtein A) in pharmaceutical formulations. The generally accepted standard is ≤ 50 ng / mg.

[0583] General technicians in the biomedical field can detect rDNA impurities in pharmaceutical preparations using reverse transcriptase PCR. The generally accepted standard is <10 ng per dose.

[0584] A person with general technical skills in the biomedical field can use RP-high performance liquid chromatography to confirm the purity of pharmaceutical formulations. For example, a person with general technical skills in the biomedical field can confirm the presence of residual free drug and free protein intermediates.

[0585] General technicians in the biomedical field can use the United States Pharmacopeia - National Formulary (USP) <85> The presence of endotoxins in pharmaceutical preparations can be determined according to the standard analytical method described in the European Pharmacopoeia, Ph. 2.6.14. The generally accepted standard is based on a dose-dependent response of < 5 EU / kg.

[0586] General technicians in the biomedical field can use the United States Pharmacopeia - National Formulary (USP) <61> Or the bioload of the active pharmaceutical ingredient as described in the European Pharmacopoeia Ph.2.6.12. The generally accepted standard is ≤ 1 CFU / 10 mL.

[0587] General technicians in the biomedical field can use the United States Pharmacopeia - National Formulary (USP) <71> Or the sterility of the pharmaceutical product as described in the European Pharmacopoeia Ph.2.6.1. The generally accepted standard is no growth.

[0588] A person with general technical skills in the biomedical field can use HPLC-ELSD methods to analyze the presence of excipients in pharmaceutical products. For example, a person with general technical skills in the biomedical field can measure the percentage of surfactants. A generally accepted standard is ±50% of the target value.

[0589] General technicians in the biomedical field can use the United States Pharmacopeia - National Formulary (USP) <787> Or, as described in the European Pharmacopoeia Ph. 2.6.19, the standard for analyzing subvisible particles in the container of a pharmaceutical product. Several recognized standards are known to those skilled in the art in the biomedical field.

[0590] General technicians in the biomedical field can use the United States Pharmacopeia - National Formulary (USP) <697> Or the extractable volume of a standard analytical pharmaceutical product as described in the European Pharmacopoeia Ph.2.6.17.

[0591] General technicians in the biomedical field can use the United States Pharmacopeia - National Formulary (USP) <1207> The standards described herein apply to container seal integrity testing (CCIT) for pharmaceutical products. Bioburden testing is typically performed only at the final stabilization time. CCIT testing is usually performed at repeated intervals, such as annually.

[0592] Statistical analysis .

[0593] Normally distributed quantitative variables were expressed as mean and SD and compared using an independent-sample t-test, as done by Zhang et al. (October 2019). For non-normally distributed variables, we used the median and interquartile range and analyzed them using the Mann-Whitney U test. Categorical data were summarized as percentages. A two-tailed p-value < 0.05 was considered statistically significant. All statistical tests were performed using SPSS version 16.0.

[0594] % mol / mol = C(free drug) / [C(free drug) + BAR × C(protein)] × 100, where:

[0595] BAR: Drug-to-Antibody Ratio

[0596] C (free drug): concentration of free drug in ADC (μM)

[0597] C (protein): Protein concentration in 1 mg / mL ADC (μM)

[0598] C (protein) = 1 / [MW (protein)]

[0599] Example

[0600] The present invention is further illustrated by non-limiting examples.

[0601] Example 1

[0602] Transient expression of the IgG4 binding site in mammalian cells

[0603] The inventors pre-inoculated and passaged Chinese hamster ovary K1 host cells. On the day of transfection, the inventors centrifuged the host cells to a specific cell density for transfection, and then added DNA and transfection reagent (polyethyleneimine) to the host cells for transfection. The transfected culture was incubated by shaking at 150 rpm.

[0604] Twenty-four hours after transfection, the temperature was changed from 36.5℃ to 33℃. Feeding was performed on days 0 and 4, and the cell culture was harvested on day 7 or when VIA < 60%.

[0605] Harvest and Clarification Centrifuge the cell culture medium at 10,000 g for 30 minutes, and then pass it through a 0.22 µm sterile filter.

[0606] Results: VHH-Cys (ABT502 and ABT503) and VHH-Fc fusion (ABT310) expression Chinese hamster ovary K1 cells were cultured to the target density in cell culture medium and transiently co-transfected with heavy and light chain vectors using a transfection reagent (polyethyleneimine). The transfected cells were cultured in shake flasks at 150 rpm for 24 hours at 36.5°C, followed by incubation at 33°C for the remaining culture time. Feeding was performed on days 0 and 4.

[0607] Example 2

[0608] Materials generation

[0609] Determination of screening conditions To optimize ligand concentrations and cell lines for selection, HEK-293T and QT6 cells were transfected in 384-well cell culture plates using plasmids encoding known ligand targets, protein A (binding antibody Fc; positive control), or separate vectors (pUC used as a negative control). After incubation, four quadruple dilutions of each ligand (starting from 20 µg / ml) were added to the transfected cells in quadruplicate, and ligand binding was detected using a single dilution of a fluorescently labeled secondary antibody in a high-throughput immunofluorescence flow cytometry assay.

[0610] Using ForeCyt ® The software (Intellicyt) determined the mean mean fluorescence intensity (MFI) of each test ligand dilution in each cell line and used Excel. ® (Microsoft) plotting. The high background rate for each assay condition was calculated as the percentage of positive events above the specified fluorescence threshold in cells transfected with the negative control. The optimal screening concentration and cell line for each test ligand were determined by the background signal (MFI) and the high background rate in the vector control.

[0611] Specific medication. Larger medication.The VHH-Fc fusion compound plus ASGPR ligand is conjugated to the Fc region via MATE. Advantages of AGT401 include the use of the MATE conjugation platform, greater stability of the unconjugated Fc fusion compound, and superior degradation depth and rate compared to bivalent nanobodies. Advantages of AGT402 include low immunogenicity and well-developed conjugation chemistry.

[0612] Specific dosage form. Smaller dosage form. VHH+ ASGPR ligands are linked via sorting enzyme-mediated linkage or C-terminal cysteine ​​conjugation. The advantages of AGT403 and AGT404 include their smaller size. VHH+ ASGPR ligands are linked via sorting enzyme-mediated linkage or C-terminal cysteine ​​conjugation.

[0613] Non-Fc fusion degrading agents showed IgG4 depletion. Fc fusion degrading agents showed IgG4 depletion at eight hours. Fc fusion degrading agents showed IgG4 depletion and rebound effect at twenty-four hours.

[0614] VHH production uses Chinese hamster ovaries as the host, achieving a good titer of 500 mg / L in Chinese hamster ovary cells. A 500 ml scale is useful because the recovery rate after purification in 500 ml is approximately 50% to 60%, yielding 100 mg of protein. There is a risk of dimerization / aggregation during production.

[0615]

[0616]

[0617] Example 3

[0618] Specific mutational strategies to reduce FcγR and C1q binding / effector function and enhance antibody FcRn binding / exposure slightly .

[0619] The inventors used a biomedically recognized method first described by the Winter group in the 1990s to mutate a VHH affinity ligand variant to insert an LALA mutation. In this example, LALA = L234A / L235A.

[0620] The inventors used a biomedically accepted method first described by the Winter group in the 1990s to mutate VHH affinity ligand variants to insert an LALA mutation, followed by an insert of a P mutation, as per biomedically accepted methods introduced by the Roche team in 2016, such as techniques for adding P329G and combining P329G with LALA. In this example, LALA / PA = L234A / L235A / P329A. LALA / PG = L234A / L235A / P329G. See Tilman Schlothauer et al., Protein Engineering, Design and Selection, Vol. 29, No. 10, October 2016, pp. 457-466. This paper shows that even LALA itself eliminates C1q binding. P329A was tested alone, and it eliminated C1q binding and reduced FcgR binding. This paper did not test P329A / LALA, but showed that P329G / LALA further reduced FcgR binding compared to LALA alone.

[0621] The inventors also mutated the VHH affinity ligand variant in other regions. N297A / Q removed the native N-linked glycosylation site in the hinge region.

[0622]

[0623] Example 4

[0624] VHH epitope on IgG4-Fc

[0625] The VHH epitope at the IgG4-Fc binding site on IgG4 was identified as being divided into two parts. Amino acids 47 to 57 are DVSQEDPEVQFN (SEQ ID NO: 14). Amino acids 106 to 114 are SNKGPSSI (SEQ ID NO: 15). For the structure of IgG4, see Davies et al., Molecular Immunology, Vol. 62, No. 1, pp. 46-53 (November 2014). The inventors superimposed the structure of the hIgG1-Fc / FcγRI complex onto the IgG4-Fc model using the Fc region. See Kiyoshi et al., Nature Communications, 6, 6866 (2015). The conclusion is that binding of the VHH domain may compete with binding of FcγRI. IgG4 has little or no affinity for other FcγRs or C1qs.

[0626] The inventors also used the Fc region to superimpose the structure of the hIgG1-FcIII peptide complex onto the IgG4-Fc model. See Delano et al., Science, 287, 1279-1283 (2000). The conclusion is that binding to the VHH domain is unlikely to compete with binding to the FcIII peptide or FcRn (which binds in the same region).

[0627] Example 5

[0628] Procedure for preparing AGN402

[0629] AGN402 (ABT310-TBT105).

[0630] BAR2 chemical formula (amino acid) :C3570H5554N950O1146S24.

[0631] Modification :pE(Q)*2,-K*2,G0F,G1F,BAR2.

[0632]

[0633] AGN402 was prepared using lysine-specific conjugation.

[0634] (1) Through UFDF (Pellicon) ® 3, 10 kDa, 0.11 m 2 The ABT310 buffer in the original buffer (20 mM histidine-acetate, 150 mM sodium chloride, pH 5.5) was replaced with 20 mM PB, pH 6.5, and subjected to 14 DV. The concentration, as determined by Nanodrop, was 16.47 mg / mL.

[0635] (2) 4000 mg of antibody was transferred to a 1 L vial (Corning, 430518) and placed in an ice bath; then conjugation buffer (20 mM PB, pH 6.5) and 10 mM TBT105 solution (tri-equivalent) were added. The total concentration of dimethyl sulfoxide in the reaction mixture was 3.74%; the reaction concentration was 10 mg / mL.

[0636] (3) Bring the reaction vessel to near ambient temperature and then incubate at 25°C. Monitor the reaction using liquid chromatography-mass spectrometry, and the BAR value reaches 2.00 after ten hours. Then place the reaction vessel at 4°C overnight (seventeen hours).

[0637] (4) Then the crude conjugate is treated with UFDF (Pellicon) ®3, 10 kDa, 2*0.11 m 2 The product was purified at 160 DV with phosphate-buffered saline, pH 7.4 (Gibco, 10010031) and 15% dimethyl sulfoxide (Sigma, D4540), followed by purification at 20 DV with phosphate-buffered saline, pH 7.4. Two batches of product were obtained, yielding 3776.88 mg of AGN402 (001) (77.30 mg / mL) and 22.44 mg of AGN402 (002) (11.22 mg / mL).

[0638] (5) Filter the purified sample using a 0.22 μm filter.

[0639] (6) Quality control tests were performed, including concentration, size exclusion chromatography-high performance liquid chromatography, hydrophobic interaction chromatography-high performance liquid chromatography, liquid chromatography-mass spectrometry, free drug level, and endotoxin level. All quality control tests yielded acceptable and expected results. The measured mass was 83.9 kDa.

[0640] The major conjugation site was also identified as K154, which was determined by peptide mapping (Thermo / Q-Exactive Orbitrap).

[0641]

[0642] Example 6

[0643] Procedure for preparing AGN508

[0644] AGN508 (ABT501-TBT103).

[0645] BAR1 chemical formula (amino acid) C671H1042N193O229S5.

[0646] Modification pE(Q),Cys,BAR1.

[0647] AGN508 is prepared using the Michael addition reaction.

[0648] (1) reductionTo a 1 L flask (Corning, 430518) containing 2000 mg of ABT501 in its original buffer (7.26 mg / mL in 20 mM histidine acetate, 150 mM sodium chloride, pH 5.5), add 10% (v / v) 1 M Tris buffer, pH 7.5, to adjust the pH of the conjugation buffer to 6-7, along with 5 mM tris(2-carboxyethyl)phosphonic acid hydrochloride solution (1.5 equivalents) and 200 mM DTPA solution. The protein concentration in the reaction was 5.1 mg / mL. The DTPA concentration in the reaction was 4 mM. The reactants were thoroughly mixed and incubated at 22°C for one hour.

[0649] (2) Adhesion Add TBT103 (10 mM in dimethyl sulfoxide) (three equivalents) to the reaction, mix thoroughly, and incubate at 22°C for another hour.

[0650] (3) Then the crude conjugate is treated with UFDF (Pellicon) ® 3, 3 kDa, 2*0.11 m 2 The sample was purified with phosphate-buffered saline, pH 7.4 (Gibco, 10010031) for 70 DV to give 1949 mg of AGN508.

[0651] (4) Filter the purified sample using a 0.22 μm filter.

[0652] (5) Quality control tests were performed, including concentration, size exclusion chromatography-high performance liquid chromatography, hydrophobic interaction chromatography-high performance liquid chromatography, liquid chromatography-mass spectrometry, free drug level, and endotoxin level. All quality control tests yielded acceptable and expected results. The measured mass was 15.6 kDa.

[0653] Example 7

[0654] Procedure for preparing AGN501

[0655] AGN501 (ABT502-TBT103).

[0656] BAR1 chemical formula (amino acid) C624H983N170O217S5.

[0657] Modification pE(Q),Cys,BAR1.

[0658] AGN501 is prepared using the Michael addition reaction.

[0659] (1) reductionTo a 1 L flask (Corning, 430518) containing 2000 mg of ABT502 in its original buffer (7.65 mg / mL in 20 mM histidine acetate, 150 mM sodium chloride, pH 5.5), add 10% (v / v) 1 M Tris buffer, 7.5 to adjust the pH of the conjugation buffer to 6-7, 5 mM tris(2-carboxyethyl)phosphonic acid hydrochloride solution (1.5 equivalents), and 200 mM DTPA solution. The protein concentration in the reaction is 5.15 mg / mL. The DTPA concentration in the reaction is 4 mM. Mix the reactants thoroughly and incubate at 22°C for one hour.

[0660] (2) Adhesion Add TBT103 (10 mM in dimethyl sulfoxide) (three equivalents) to the reaction, mix thoroughly, and incubate at 22°C for another hour.

[0661] (3) Then the crude conjugate is treated with UFDF (Pellicon) ® 3, 3 kDa, 2*0.11 m 2 The solution was purified with phosphate-buffered saline, pH 7.4 (Gibco, 10010031) for 70 DV to give 1717.8 mg of AGN501.

[0662] (4) Filter the purified sample using a 0.22 μm filter.

[0663] (5) Quality control tests were performed, including concentration, size exclusion chromatography-high performance liquid chromatography, hydrophobic interaction chromatography-high performance liquid chromatography, liquid chromatography-mass spectrometry, free drug level, and endotoxin level. All quality control tests yielded acceptable and expected results. The measured mass was 14.5 kDa.

[0664] Example 8

[0665] Procedure for preparing AGN502

[0666] AGN502 (ABT503-TBT103).

[0667] BAR1 chemical formula (amino acid) C627H987N170O219S5.

[0668] Modification pE(Q),Cys,BAR1.

[0669] AGN502 is prepared using the Michael addition reaction.

[0670] (1) reductionTo a 1 L flask (Corning, 430518) containing 2000 mg of ABT503 in its original buffer (7.41 mg / mL in 20 mM histidine acetate, 150 mM sodium chloride, pH 5.5), add 10% (v / v) 1 M Tris buffer, pH 7.5, to adjust the pH of the conjugation buffer to 6-7, along with 5 mM tris(2-carboxyethyl)phosphonic acid hydrochloride solution (1.5 equivalents) and 200 mM DTPA solution. The protein concentration in the reaction was 5.04 mg / mL. The DTPA concentration in the reaction was 4 mM. The reactants were thoroughly mixed and incubated at 22°C for one hour.

[0671] (2) Adhesion Add TBT103 (10 mM in dimethyl sulfoxide) (three equivalents) to the reaction, mix thoroughly, and incubate at 22°C for another hour.

[0672] (3) Then the crude conjugate is treated with UFDF (Pellicon) ® 3, 3 kDa, 2*0.11 m 2 The solution was purified by phosphate-buffered saline, pH 7.4 (Gibco, 10010031) for 70 DV to give 2008 mg of AGN502.

[0673] (4) Filter the purified sample using a 0.22 μm filter.

[0674] (5) Quality control tests were performed, including concentration, size exclusion chromatography-high performance liquid chromatography, hydrophobic interaction chromatography-high performance liquid chromatography, liquid chromatography-mass spectrometry, free drug level, and endotoxin level. All quality control tests yielded acceptable and expected results. The measured mass was 14.6 kDa.

[0675] Example 9

[0676] Procedure for preparing AGN509

[0677] AGN509 (ABT504-TBT103)

[0678] BAR1 chemical formula (amino acid) C660H1025N188O223S5

[0679] Modification pE(Q),Cys,BAR1.

[0680] AGN509 is prepared using the Michael addition reaction.

[0681] (1) reductionTo a 1 L flask (Corning, 430518) containing 1900 mg of ABT504 in its original buffer (7.78 mg / mL in 20 mM histidine acetate, 150 mM sodium chloride, pH 5.5), add 10% (v / v) 1 M Tris buffer, pH 7.5, to adjust the pH of the conjugation buffer to 6-7, along with 5 mM tris(2-carboxyethyl)phosphonic acid hydrochloride solution (1.5 equivalents) and 200 mM DTPA solution. The protein concentration in the reaction was 5.30 mg / mL. The DTPA concentration in the reaction was 4 mM. The reactants were thoroughly mixed and incubated at 22°C for one hour.

[0682] (2) Adhesion Add TBT103 (10 mM in dimethyl sulfoxide) (three equivalents) to the reaction, mix thoroughly, and incubate at 22°C for another hour.

[0683] (3) Then the crude conjugate is treated with UFDF (Pellicon) ® 3, 3 kDa, 2*0.11 m 2 The phosphate-buffered saline solution at pH 7.4 (Gibco, 10010031) was purified for 70 DV to give 1896.35 mg of AGN509.

[0684] (4) Filter the purified sample using a 0.22 μm filter.

[0685] (5) Quality control tests were performed, including concentration, size exclusion chromatography-high performance liquid chromatography, hydrophobic interaction chromatography-high performance liquid chromatography, liquid chromatography-mass spectrometry, free drug level, and endotoxin level. All quality control tests yielded acceptable and expected results. The measured mass was 15.3 kDa.

[0686] Example 10

[0687] Procedure for preparing AGN401

[0688] AGN401 (ABT306-TBT103).

[0689] BAR1 chemical formula (amino acid) C719H1115N202O244S5.

[0690] Modification pE(Q), Cys, O-glycan, a payload.

[0691]

[0692] AGN401 is prepared using the Michael addition reaction.

[0693] (1) reduction To a 150 mL flask (Corning, 430281) containing 150 mg of ABT306 in its original buffer (3.59 mg / mL in 20 mM histidine acetate, 150 mM sodium chloride, pH 5.5), add 10% (v / v) 1 M Tris buffer, pH 7.5, to adjust the pH of the conjugation buffer to 6-7, along with 5 mM tris(2-carboxyethyl)phosphonate hydrochloride solution (1.2 equivalents) and 200 mM EDTA solution. The protein concentration in the reaction was 2.94 mg / mL. The EDTA concentration in the reaction was 2 mM. The reactants were thoroughly mixed and incubated at 22°C for three hours.

[0694] (2) Adhesion Add TBT103 (10 mM in dimethyl sulfoxide) (three equivalents) to the reaction, mix thoroughly, and incubate at 22°C for another hour.

[0695] (3) Then use Amicon to compost the coarse compound. ® (Millipore, 10 kDa, 15 mL) was purified with phosphate-buffered saline at pH 7.4 (Gibco, 10010031) to give 135.24 mg of AGN401.

[0696] (4) Filter the purified sample using a 0.22 μm filter.

[0697] (5) Quality control tests were performed, including concentration, size exclusion chromatography-high performance liquid chromatography, hydrophobic interaction chromatography-high performance liquid chromatography, liquid chromatography-mass spectrometry, free drug level, and endotoxin level. All quality control tests yielded acceptable and expected results. The measured mass was 19.6 kDa.

[0698] Example 11

[0699] Procedure for preparing AGN403

[0700] AGN403 (ABT304-TBT106).

[0701] BAR1 chemical formula C676H1068N188O235S6.

[0702] Modification pE(Q), BAR1.

[0703] AGN403 is prepared using ligation catalyzed by sorting enzymes.

[0704] (1) To a 150 mL flask (Corning, 430281) containing 300 mg ABT304 in the original buffer (6.30 mg / mL in 20 mM histidine acetate, 150 mM sodium chloride, pH 5.5), add 5.08 mg / mL sorting enzyme A (PT-0008, 0.01 equivalents), conjugation buffer (50 mM HEPES, 150 mM sodium chloride, 10% glycerol (v / v), pH 7.5), and TBT106 (40 mM dimethyl sulfoxide solution) (eight equivalents) to the reaction mixture, mix thoroughly, and incubate overnight (17 hours) at 4°C. The concentration of ABT304 in the reaction is 3 mg / mL.

[0705] (2) The crude conjugate was purified by AKTA-purified chromatography (column: HisTrap excel, 1 mL; equilibration buffer: 20 mM sodium phosphate, 500 mM sodium chloride, 10 mM imidazole, pH 7.4; elution buffer: 0.2 M sodium hydroxide; flow rate: 1 mL / min). The conjugate was flowed through during loading. His-tagged sorting enzymes and His-tagged unconjugated peptides were eluted with 0.2 M sodium hydroxide. The inventors pooled the streams and used Amicon... ® (Milipore, 10 kDa, 15 mL) Replace the buffer with phosphate-buffered saline, 2 mM EDTA, pH 7.4, and perform 30 DV.

[0706] (3) Filter the purified sample using a 0.22 μm filter.

[0707] (4) Quality control tests were performed, including concentration, size exclusion chromatography-high performance liquid chromatography, hydrophobic interaction chromatography-high performance liquid chromatography, liquid chromatography-mass spectrometry, free drug level, and endotoxin level. All quality control tests yielded acceptable and expected results. The measured mass was 15.8 kDa.

[0708] Example 12

[0709] Procedure for preparing AGN404

[0710] AGN404 (ABT305-TBT106).

[0711] BAR1 chemical formula C657H1040N182O229S4.

[0712] Modification pE(Q), BAR1.

[0713] AGN404 is prepared using ligation catalyzed by sorting enzymes.

[0714] (1) To a 150 mL flask (Corning, 430281) containing 300 mg ABT305 in the original buffer (5.03 mg / mL in 20 mM histidine acetate, 150 mM sodium chloride, pH 5.5), add 5.08 mg / mL sorting enzyme A (PT-0008, 0.01 equivalents), conjugation buffer (50 mM HEPES, 150 mM sodium chloride, 10% glycerol (v / v), pH 7.5), and TBT106 (40 mM dimethyl sulfoxide solution) (eight equivalents) to the reaction mixture, mix thoroughly, and incubate overnight (17 hours) at 4°C. The concentration of ABT305 in the reaction is 3 mg / mL.

[0715] (2) The crude conjugate was purified by AKTA-purified chromatography (column: HisTrap excel, 1 mL; equilibration buffer: 20 mM sodium phosphate, 500 mM sodium chloride, 10 mM imidazole, pH 7.4; elution buffer: 0.2 M sodium hydroxide; flow rate: 1 mL / min). The conjugate was flowed through during loading. His-tagged sorting enzymes and His-tagged unconjugated peptides were eluted with 0.2 M sodium hydroxide. The streams were pooled and purified using Amicon chromatography. ® (Milipore, 10 kDa, 15 mL) Replace the buffer with phosphate-buffered saline, 2 mM EDTA, pH 7.4, and perform 30 DV.

[0716] (3) Filter the purified sample using a 0.22 μm filter.

[0717] (4) Quality control tests were performed, including concentration, size exclusion chromatography-high performance liquid chromatography, HIC-high performance liquid chromatography, liquid chromatography-mass spectrometry, free drug level, and endotoxin level. All quality control tests yielded acceptable and expected results. The measured mass was 15.2 kDa.

[0718] Example 13

[0719] Preparation procedure for building block #1

[0720] Preparation of intermediate 2

[0721]

[0722] Under a nitrogen atmosphere, Pd(OH)₂ (8.00 g, 56.9 mmol, 0.44 equivalent) and hydrochloric acid (2 M, 64.4 mL, 1.00 equivalent) were added to a mixture of reactant 1 (20.0 g, 128 mmol, 1.00 equivalent) in ethanol (400 mL). The mixture was stirred at 25 °C for twenty hours under a hydrogen atmosphere (50 psi). Thin-layer chromatography (DCM: methanol = 10:1) showed that reactant 1 was consumed and formed a main spot (R). f = 0.5). The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The crude product was ground with ethyl acetate (100 mL) at 25 °C for two hours to give intermediate 2 (18.0 g, 92.0 mmol, 71.3% yield, 100% purity, HCl salt) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ = 8.51– 7.71 (m, 3H), 7.21 - 7.17 (m, 1H), 6.93 - 6.88 (m, 1H), 3.94 (s, 2H).

[0723] Preparation of intermediate 4

[0724]

[0725] NMM (15.9 g, 157 mmol, 17.3 mL, 2.00 equivalent) and intermediate 2 (13.1 g, 67.2 mmol, 0.85 equivalent, HCl salt) were added to a solution of reactant 3 (40.0 g, 78.6 mmol, 1.00 equivalent) in dimethylformamide (200 mL). The mixture was stirred at 25 °C for two hours. Liquid chromatography-mass spectrometry (LC-MS) showed complete consumption of intermediate 2. A main peak with the expected mass was detected (retention time = 0.48 min) (MS calculated value: 552.2, MS observed value: [M-tBu+ H)). + = 497.1). The residue was diluted with water (800 mL) and extracted with ethyl acetate (1500 mL) (500 mL * 3). The combined organic layers were washed with 500 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was ground with ethyl acetate (1000 mL) at 25 °C for four hours. Intermediate 4 (19.0 g, 33.1 mmol, 42.1% yield, 96.4% purity) was obtained as a yellow solid. The retention time by liquid chromatography-mass spectrometry was 0.48 min, and the calculated MS value was 552.2. The MS measured value was [M - tBu+H]. += 497.1.

[0726] Preparation of building block #1

[0727]

[0728] Trifluoroacetic acid (154 g, 1.35 mol, 100 mL, 39.2 equivalents) was added to a solution of intermediate 4 (19.0 g, 34.3 mmol, 1.00 equivalents) in DCM (100 mL). The mixture was stirred at 25 °C for two hours. Liquid chromatography-mass spectrometry (LC-MS) results showed complete consumption of intermediate 4 and detection of a main peak with the expected mass (RT = 0.41 min) (MS calculated value was 496.1, MS observed value was [M+H]). + = 497.1). The mixture solution was concentrated under reduced pressure to obtain the residue. The residue was purified by reversed-phase HPLC (trifluoroacetic acid conditions) to give building block #1 as a white solid (9.10 g, 17.9 mmol, 52.1% yield, 97.9% purity). Liquid chromatography-mass spectrometry retention time = 0.41 min, calculated MS value 496.1, measured MS value [M+Na] + = 519.1. HPLC shows a retention time of 2.95 min and a purity of 97.9%. SFC (EC6536-156-P1C) shows a retention time of 1.58 min and a value of 97.8%. 1 H NMR (400 MHz, DMSO-d6) δ = 13.60-11.82 (m, 1H), 10.26 ( s, 1H), 8.34 ( t, J = 5.6 Hz, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.70 (d, J = 7.6 Hz, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.48-7.37 (m, 2H), 7.36-7.27(m, 2H), 6.92 ( t, J = 7.6 Hz, 1H), 6.70 ( t, J = 7.6 Hz, 1H), 4.46-4.33 (m,1H), 4.29-4.19 (m, 5H), 2.69-2.61 (m, 1H), 2.52 (s, 1H).

[0729] Example 14

[0730] Procedure for preparing TBT105

[0731] Preparation of intermediate 7

[0732]

[0733]

[0734]

[0735] This chemical compound was synthesized using standard Fmoc chemistry (CTC resin).

[0736] (1) Resin preparation Add N,N-diisopropylethylamine (4.00 equivalents) dropwise to a container containing a DCM (50 mL) solution of CTC resin (22.7 g, 10.0 mmol, 0.55 mmol / g) and Fmoc-Thr(tBu)-OH (3.97 g, 10.0 mmol, 1.00 equivalents) and mix for two hours at 25°C under bubbling nitrogen. Then add methanol (22.7 mL) and bubble with nitrogen for another thirty minutes. Wash the resin with dimethylformamide (100 mL) * 5, then add a 20% piperidine solution in dimethylformamide (100 mL) and bubble with nitrogen at 25°C for thirty minutes to deprotect the Fmoc. Filter the mixture. Wash the resin with dimethylformamide (100 mL) * 5 and proceed to the next step.

[0737] (2) Couplet A solution of Fmoc-Cys(Trt)-OH (17.5 g, 30.0 mmol, 3.00 equivalents) and HBTU (10.8 g, 28.5 mmol, 2.85 equivalents) in dimethylformamide (500 mL) was added to the resin under bubbling nitrogen. Then, N,N-diisopropylethylamine (6.00 equivalents) was added dropwise to the mixture, and the mixture was bubbled under nitrogen at 25°C for 30 minutes. The coupling reaction was monitored using a ninhydrin test; a colorless result indicated completion of the coupling reaction. The resin was then washed with dimethylformamide (300 mL) * 5.

[0738] (3) Deprotection Add a 20% piperidine solution in 100 mL of dimethylformamide to the resin and bubble the mixture under nitrogen at 25°C for 30 minutes. Then wash the resin with 100 mL of dimethylformamide.

[0739] (4) Repeat steps 2 and 3 to perform the following amino acid extension. See numbers 3 to 15 in Table 17.

[0740] (5) After all steps are completed, the resin is washed with dimethylformamide (100 mL) * 5 and methanol (100 mL) * 5, and then dried under reduced pressure to obtain resin-bound peptide intermediate 5 (CTC resin, 10.0 mmol).

[0741]

[0742] Peptide cleavage and cyclization .

[0743] (1) At 25°C, the cleavage solution (TFA / TIS / water, 95 / 2.5 / 2.5, v / v / v, 420 mL) was added to a flask containing a resin-bound peptide (CTC resin, 42 g, 10.0 mmol) with side chain protection and stirred for two hours.

[0744] (2) After filtration, collect the filtrate.

[0745] (3) The filtrate was precipitated with cold isopropyl ether (2.5 L). After filtration, the solid was washed twice with isopropyl ether (2.5 L) and the crude peptide was dried under reduced pressure for two hours to obtain intermediate 6 (16.5 g, crude product) as a white solid.

[0746] Intermediate 6 (16.5 g, crude) was added dropwise to a mixture of HOAc / MeCN / water (4 / 3 / 3, v / v / v, 8 L) with 0.1 M I2 / AcOH until the yellow color persisted, and then the mixture was stirred at 25 °C for five minutes. The mixture was quenched by adding dropwise 0.1 M Na2S2O3 aqueous solution until the yellow color disappeared. After filtration, the filtrate was purified by preparative high-performance liquid chromatography (A: 0.075% TFA / water, B: MeCN) and then lyophilized to give intermediate 7 (6 g, 90.0% purity, 32.43% yield) as a white solid. Liquid chromatography-mass spectrometry: retention time = 0.960 min, MS calculated value: M av = 1851.01, Mass observation: [M + 2H] 2+ =925.90, [M + H] + =1852.01.

[0747] Preparation of intermediate 8

[0748]

[0749] At 0 °C, a mixture of target A001 (4.09 g, 1.00 equivalent) and N,N-diisopropylethylamine (1.283 g, 1.307 mL, 3 equivalent) in 50 mL of dimethylformamide was added to a solution of bis-PEG4-TFP (5.18 g, 3.00 equivalent) in dimethylformamide (25 mL). The resulting reaction was stirred at 0 °C for five minutes. After completion by liquid chromatography-mass spectrometry (LC-MS), the mixture was directly injected into a reversed-phase column and purified by preparative high-performance liquid chromatography (A: 0.075% TFA / water, B: MeCN), followed by lyophilization to give intermediate 8 (2.9 g, purity 95.0%, yield 50.7%) as a colorless oil. LC-MS retention time = 0.709 min, MS calculated value: M av = 1821.81, Mass observation: [M+16 + 2H] 2+ =911.60.

[0750] Preparation of TBT105

[0751]

[0752] N,N-diisopropylethylamine (358.73 mg, 2.77 mmol, 459.92 μL, 2.00 equivalent) was added to a solution of intermediate 8 (3.34 g, 1.80 mmol, 1.30 equivalent) in dimethylformamide (35 mL). The mixture was stirred at 20 °C for one hour. After monitoring by liquid chromatography-mass spectrometry, the mixture was acidified to pH = 5 with 1 M hydrochloric acid. The mixture was directly purified by preparative high-performance liquid chromatography (A: 0.075% TFA / water, B: MeCN) and then lyophilized to give TBT105 as a white solid (1.52 g, 96.0% purity, 29.98% yield). Liquid chromatography-mass spectrometry retention time = 0.817 min, MS calculated value: M av = 3505.75, Mass observation: [M + 3H] 3+ =1169.20.

[0753] Example 15

[0754] Procedure for preparing TBT103

[0755]

[0756]

[0757] 2,6-Dimethylpyridine (3.37 g, 3.8 mL, 31.49 mmol, 2.0 equivalent) was added to a solution of target A001 (25 g, 15.74 mmol, 1.0 equivalent) in NMP (125 mL, 5 V). Then, Mal-PFP ester (5.3 g, 15.81 mmol, 1.0 equivalent) was added. The solution was stirred at 25 °C for one hour. After completion, monitored by liquid chromatography-mass spectrometry, trifluoroacetic acid was added to quench the reaction until pH = 6. The mixture was added dropwise to methyl tert-butyl ether (3 L) and the supernatant was decanted, yielding the residue. The residue was purified twice by preparative high-performance liquid chromatography (HPLC) (column: Phenomenex luna c18 250 mm * 100 mm * 15 μm; mobile phase: [A: water (0.2% FA); B: ACN]; B%: 0.00%–20.00%, 21.00 min), and then lyophilized to give TBT103 as a white solid (9.5 g, 98.1% purity, 34% yield). HPLC-MS retention time = 17.129 min, MS calculated value: M av = 1548.59, Mass observation: [M + H] + =1549.50.

[0758] Example 16

[0759] Procedure for preparing TBT106

[0760]

[0761]

[0762] Preparation of intermediate 9

[0763]

[0764] Peptides were synthesized using standard Fmoc chemical synthesis.

[0765] (1) Resin preparation Add dimethylformamide (100.0 mL) to a container containing Rink Amide MBHA resin (sub: 0.27 mmol / g, 2.00 mmol, 7.5 g).

[0766] (2) Deprotection Add 20% piperidine / dimethylformamide (200.0 mL) and mix for 30 minutes at room temperature (25°C). Monitor the deprotection reaction using the ninhydrin test; if it turns blue or brownish-red, the reaction is complete. Then wash the resin with dimethylformamide (200.0 mL) * 5.

[0767] (3) Couplet A 100.0 mL solution of Fmoc-amino acids in dimethylformamide was added to the resin under nitrogen bubbling, followed by the addition of activation buffer to the resin at room temperature (25°C) under nitrogen bubbling. One hour. The coupling reaction is monitored by a ninhydrin test; if it shows a colorless result, the coupling is complete. The resin is then washed with dimethylformamide (200.0 mL) * 5.

[0768] (4) Repeat steps 2 to 3 for all other amino acids: (numbers 1 to 3 in Table 18). As a final step, wash the resin with methanol (200.0 mL) * 3 and dry it by bubbling with nitrogen for three hours.

[0769]

[0770] Peptide cleavage and cyclization .

[0771] (1) At 25°C, the cutting solution (TFA / TIS / water, 95 / 2.5 / 2.5, v / v / v, 90 mL) was added to a flask containing a resin-bound peptide (Rink Amide MBHA resin, 9 g, 2.0 mmol) with side chain protection and stirred for two hours.

[0772] (2) After filtration, collect the filtrate.

[0773] (3) The filtrate was precipitated with cold isopropyl ether (450 mL). After filtration, the solid was washed twice with isopropyl ether (450 mL), and the crude peptide was dried under reduced pressure for two hours to obtain intermediate 1 (1 g, crude product) as a white solid. The crude peptide was purified by preparative high performance liquid chromatography (A: 0.1% TFA / water, B: MeCN) and then lyophilized to obtain intermediate 9 (520 mg, 58.23% yield, 89% purity) as a white solid.

[0774] Preparation of TBT106

[0775]

[0776]

[0777] At 25°C and under nitrogen, CuSO4 (0.4 M, 1.68 mL, 1 equivalent) was added dropwise to an aqueous (5 mL) solution of sodium ascorbate (399.32 mg, 2.02 mmol, 3 equivalents). The mixture turned brown, and then THPTA (291.94 mg, 671.89 μmol, 1 equivalent) was added to obtain a clear solution A. Solution A was added to an aqueous (10 mL) solution B containing target A043 (1.084 g, 671.892 μmol, 1 equivalent) and intermediate 9 (300 mg, 671.892 μmol, 1 equivalent) at 25°C and under nitrogen. The mixture was stirred at 25°C for 0.5 hours. After monitoring by liquid chromatography-mass spectrometry, the crude solution was purified by preparative high-performance liquid chromatography (A: 0.1% TFA / water, B: MeCN) to obtain the crude product. Further purification was performed by preparative high-performance liquid chromatography (A: 0.5% AcOH / water, B: MeCN), followed by lyophilization to obtain TBT106 as a white solid (590 mg, 96.5% purity, 36% yield). Liquid chromatography-mass spectrometry retention time = 0.55 min, MS calculated value: M av =June 2010, Mass observation: [M + H] + =2011.1.

[0778] Example 17

[0779] Chemical synthesis. Procedure for preparing intermediate A001A of TBT103. .

[0780] Preparation of intermediate 2

[0781]

[0782] At 0 °C, a solution of 1 (34.2 g, 200 mmol, 1.00 equivalent) in 2-methyltetrahydrofuran (160 mL) was added to a solution of 1a (60.0 g, 400 mmol, 2.00 equivalent) in 2-methyltetrahydrofuran (450 mL). The mixture was stirred at 25 °C for two hours. Thin-layer chromatography (DCM: methanol = 20:1, R...) was then performed. f = 0.70) indicates the reaction is complete, and a new major spot of low polarity is detected. HCl / EA (1 N, 27.0 mL) was added to the reaction mixture and stirred for 30 minutes. The white precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure to give intermediate 2 (crude product, 105.0 g, 370.6 mmol), a yellow oily substance. Liquid chromatography-mass spectrometry retention time = 0.797 min, MS calculated value: 283.14, observed mass: [M+ Na] + = 306.1.1 H NMR (400 MHz, DMSO-d6) δ ppm 7.23-7.41 (m, 5 H), 5.01 (s, 2H), 4.60 (br s, 1 H), 3.45-3.52 (m, 6 H), 3.38-3.43 (m, 5 H), 3.14 (q, J =5.94 Hz, 2 H), 2.53-2.55 (m, 1 H).

[0783] Preparation of intermediate 3

[0784]

[0785] TMSOTf (85.6 g, 385 mmol, 1.50 equivalent) was added to a solution of 2a (100.0 g, 257 mmol, 1.00 equivalent) in DCE (500 mL) and stirred at 60 °C for two hours. The reaction was then cooled to room temperature (25 °C) and stirred for another hour. A mixture of intermediate 2 (80.0 g, 282 mmol, 1.10 equivalent) and 4 Å powdered molecular sieve (50.0 g) in DCE (500 mL) was added to the reaction. The resulting mixture was stirred under a N2 atmosphere for thirty minutes. Then, a solution of intermediate 2a (100.0 g, 257 mmol, 1.00 equivalent) in DCE was added dropwise to the mixture at 0 °C. The mixture was stirred at 25 °C under a N2 atmosphere for sixteen hours. Thin-layer chromatography (DCM: methanol = 10:1, R...) was performed. f = 0.42) indicates that intermediate 2a was completely consumed, and a major new spot with high polarity was detected. The reaction mixture was filtered and washed with saturated NaHCO3 (500 mL), water (500 mL), and brine (500 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, PE:EA = 3:1 to 1:6, then DCM:methanol = 20:1) to give intermediate 3 (90.0 g, 146.9 mmol, 91.6% purity, 57.2% yield) as a yellow oil. Liquid chromatography-mass spectrometry was performed at room temperature = 0.860 min, MS calculated value: 612.25, mass observation: [M+H] + = 613.2. 1HNMR (400 MHz, DMSO-d6) δ ppm 7.80 (d, J = 9.03 Hz, 1 H), 7.24-7.39 (m, 6 H), 5.22 (d, J = 3.51 Hz, 1 H), 4.95-5.05 (m, 3 H), 4.53-4.59 (m, 1 H), 3.99-4.06(m, 3 H), 3.84-3.92 (m, 1 H), 3.73-3.82 (m, 1 H), 3.55-3.61 (m, 1 H), 3.45-3.53 (m, 7 H), 3.41 (t, J = 5.90 Hz, 2 H), 3.11-3.18 (m, 3 H), 2.10 (s, 3 H), 1.99 (s, 3 H), 1.89 (s, 3 H), 1.77 (s, 3 H).

[0786] Preparation of intermediate 4

[0787]

[0788] Tetrahydrofuran (180 mL) was slowly added to a reaction flask containing Pd / C (9.00 g, 10% purity) purged three times with argon. Then, under nitrogen atmosphere, a solution of trifluoroacetic acid (16.7 g, 147 mmol, 1.00 equivalent) and intermediate 3 (90.0 g, 147.0 mmol, 1.00 equivalent) in tetrahydrofuran (720 mL) was slowly added to the reaction mixture. The reaction mixture was degassed and purged three times with nitrogen and hydrogen, then stirred at 25 °C for three hours under a hydrogen atmosphere (40 psi). Thin-layer chromatography (DCM:methanol = 10:1, R...) was performed. f = 0.20) indicates that intermediate 3 was completely consumed, and a major new spot with high polarity was detected. The reaction mixture was dissolved in tetrahydrofuran (100 mL), carefully filtered through silica at a nitrogen atmosphere, the filter cake was washed with tetrahydrofuran (100 mL * 2), and the filtrate was concentrated under reduced pressure to give the residue. The residue was diluted with water (1000 mL), washed with DCM (300 mL * 3), and the aqueous layer was lyophilized to give intermediate 4 as a white solid (80.0 g, 139.0 mmol, 95.1% purity, 91.8% yield, trifluoroacetate). Liquid chromatography-mass spectrometry showed a retention time of 0.484 min, MS calculated value: 478.22, and mass observation: [M + H]. + = 478.9. 1H NMR (400 MHz, DMSO-d6) δ ppm7.91 (br t, J = 9.03 Hz, 4 H), 5.21 (d, J = 3.26 Hz, 1 H), 4.96 (dd, J =11.17, 3.39 Hz, 1 H), 4.54 (d, J = 8.53 Hz, 1 H), 3.98-4.08 (m, 3 H), 3.85-3.93 (m, 1 H), 3.75-3.84 (m, 1 H), 3.59 (br t, J = 5.14 Hz, 3 H), 3.50-3.56(m, 6 H), 2.98 (br s, 2 H), 2.10 (s, 3 H), 2.00 (s, 3 H), 1.89 (s, 3 H), 1.78 (s, 3 H).

[0789] Preparation of intermediate 6

[0790]

[0791] A sodium hydroxide aqueous solution (5.0 M, 9.91 mL, 0.10 equivalent) was added dropwise over five minutes to a mixture of 5 (60.0 g, 495.0 mmol, 1.00 equivalent) in dimethyl sulfoxide (166 mL). After addition, the mixture was stirred at 0 to 15 °C for 5 minutes, and then 5a (254.0 g, 1.98 mol, 287 mL, 4.00 equivalent) was added dropwise to the reaction mixture at 20 °C. The resulting mixture was stirred at 25 °C for sixteen hours. Thin-layer chromatography (DCM: methanol = 10:1, R...) was performed. f = 0.7) indicates that intermediate 5 was completely consumed, and a major new spot with low polarity was detected. The resulting reaction mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in ethyl acetate (400 mL), quenched by adding water (400 mL), and extracted with ethyl acetate (400 mL * 3). The combined organic layers were washed with brine (300 mL * 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give intermediate 6 as a colorless oil (100.0 g, 197.8 mmol, 96.0% purity, 40.0% yield). 1 H NMR (400 MHz, DMSO-d6) δ ppm 3.51-3.61 (m, 7 H), 3.17 (s, 5 H), 2.39 (t, J = 6.02 Hz, 6 H), 1.40 (s, 27 H).

[0792] Preparation of intermediate 7

[0793]

[0794] HOBt (10.7 g, 79.1 mmol, 1.00 equivalent) was added to a solution of intermediate 6 (40.0 g, 79.1 mmol, 1.00 equivalent) in MeCN (400 mL). Then 6a (16.5 g, 79.1 mmol, 1.00 equivalent) and DCC (16.3 g, 79.1 mmol, 1.00 equivalent) were added. The reaction mixture was stirred at 25 °C for sixteen hours. TLC (PE:EA = 1:1, R) f = 0.80) indicates that intermediate 6 was completely consumed, and a major new spot with low polarity was detected. MeCN was evaporated to give the residue. The residue was purified by column chromatography (SiO2, PE:EA = 10:1 to 1:1) to give intermediate 7 as a white solid (40.0 g, 57.4 mmol, 82.9% purity, 72.5% yield). Liquid chromatography-mass spectrometry showed a retention time of 1.151 min, calculated MS value: 696.38, and observed mass: [M+H]. + = 697.3, [M + Na] + = 719.3. 1 H NMR (400MHz, DMSO-d6) δ ppm 7.26-7.40 (m, 6 H), 7.06 (s, 1 H), 5.03 (s, 2 H), 3.49-3.61 (m, 14 H), 2.39 (br t, J = 6.02 Hz, 6 H), 1.40 (s, 27H).

[0795] Preparation of intermediate 8

[0796]

[0797] The solution of intermediate 7 (30.0 g, 43.0 mmol, 1.00 equivalent) in HCOOH (300 mL) was stirred at 25 °C for sixteen hours. Thin-layer chromatography (PE:EA = 1:1, R...) f= 0.04) indicates that intermediate 7 was completely consumed, and a major new spot with high polarity was detected. The solvent was evaporated under reduced pressure, followed by co-evaporation with toluene (50 mL * 3) under reduced pressure and drying under reduced pressure to give the residue. The residue was purified by preparative high performance liquid chromatography (A: 0.1% FA conditions / water, B: MeCN) to give intermediate 8 (20.0 g, 37.8 mmol, 98.2% purity, 87.9% yield). 1 ¹H NMR (400 MHz, DMSO-d6) δ ppm 12.17 (br s, 3 H), 7.26–7.43 (m, 6 H), 7.06 (s, 1 H), 5.02 (s, 2 H), 3.49–3.65 (m, 14 H), 2.42 (br t, J = 6.27 Hz, 6 H). Liquid chromatography-mass spectrometry (LC-MS) showed a retention time of 0.790 min, MS calculated value: 528.20, and observed mass: [M + H]. + = 529.2.

[0798] Preparation of intermediate 9 .

[0799]

[0800] To a stirred solution of intermediate 8 (20.0 g, 37.8 mmol, 1.00 equivalent) and intermediate 4 (78.5 g, 132 mmol, 3.50 equivalent, TFA salt) in dimethylformamide (400 mL), HOBT (20.4 g, 151 mmol, 4.00 equivalent), EDCI (29.0 g, 151 mmol, 4.00 equivalent), and N,N-diisopropylethylamine (22.0 g, 170 mmol, 4.50 equivalent) were added sequentially. The reaction mixture was stirred at 25 °C for two hours. Thin-layer chromatography (DCM: methanol = 10:1, R...) was performed. f = 0.4) indicates that intermediate 8 was completely consumed, and a major new spot with high polarity was detected. The reaction mixture was slowly poured into a stirred, cold 0.5 mol / L hydrochloric acid solution (900 mL) and stirred for ten minutes. A white precipitate formed and was filtered. The aqueous phase was extracted twice with DCM (600 mL * 2). The combined organic layers were washed with 5% NaHCO3 (450 mL), dried over Na2SO4, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, DCM: methanol = 100:1 to 5:1) to give intermediate 9 as a white solid (58.0 g, 30.4 mmol, 82.7% purity, 80.3% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.92 (br t, J = 5.14 Hz, 3 H), 7.81 (d, J = 9.03 Hz, 3 H), 7.28-7.39 (m, 6 H), 7.13 (s, 1 H), 5.21 (d, J = 3.26 Hz, 3 H), 5.02 (s, 2 H), 4.97 (dd, J = 11.17, 3.39 Hz, 3 H), 4.54 (d, J = 8.53 Hz, 3 H), 4.03 (s, 9H), 3.84-3.92 (m, 3 H), 3.75-3.81 (m, 3 H), 3.45-3.61 (m, 37 H), 3.39 (br s, 3 H), 3.18–3.23 (m, 6 H), 2.30 (br t, J = 6.15 Hz, 6 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.77 (s, 9 H). Liquid chromatography-mass spectrometry retention time = 3.455 min, MS calculated value: 1908.81, observed mass: [M + 2H] 2+ = 955.7.

[0801] Preparation of intermediate 10 .

[0802]

[0803] A 500 mL round-bottom flask was purged three times with argon, and dry Pd / C (1.50 g, 1.41 mmol, 10% purity, 1.00 equivalent) was carefully added. Tetrahydrofuran (150.0 mL) was then added to completely permeate the Pd / C, followed by the slow addition of a tetrahydrofuran (75 mL) solution of intermediate 9 (15.0 g, 7.85 mmol, 1.00 equivalent) and trifluoroacetic acid (895 mg, 7.85 mmol, 583 μL, 1.00 equivalent) under an argon atmosphere. The resulting mixture was degassed and purged three times with hydrogen. The mixture was then stirred at 25 °C for three hours under a hydrogen atmosphere (15 psi). The reaction was monitored by liquid chromatography-mass spectrometry, showing the desired mass (a desired main peak was detected). The reaction mixture was carefully filtered through silica clay under a nitrogen atmosphere, and the filter cake was washed with tetrahydrofuran (100 mL x 2). Then, water was immediately added to the filter cake. The organic layer was concentrated under reduced pressure to give intermediate 10 (13.0 g, 6.54 mmol, 83.2% yield, 99.7% purity, trifluoroacetate), which was a white solid.1 H NMR(400 MHz, DMSO-d6) δ ppm 7.89-7.99 (m, 5 H), 7.82 (d, J = 9.3 Hz, 3 H), 7.74(s, 1 H), 7.14-7.28 (m, 1 H), 5.22 (d, J = 3.3 Hz, 3 H), 4.97 (dd, J = 11.3,3.4 Hz, 3 H), 4.54 (d, J = 8.5 Hz, 3 H), 3.84-3.93 (m, 3 H), 3.76-3.82 (m, 3H), 3.47-3.61 (m, 43 H), 3.21 (q, J = 5.8 Hz, 6 H), 2.29-2.34 (m, 6 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 8 H), 1.77 (s, 9 H). Liquid chromatography-mass spectrometry (LC-MS) showed a retention time of 1.333 min, calculated MS value of 1774.7, and measured value of [M + 2H]. 2+ = 888.6.

[0804] Preparation of intermediate target A001A

[0805]

[0806] At 0 °C, NaOMe (5.4 M, 5.01 mL, 4.26 equivalents) was added to a solution of intermediate 10 (12.0 g, 6.35 mmol, 1.00 equivalent, trifluoroacetic acid) in methanol (120.0 mL). The mixture was stirred at 0 °C for 0.5 h. The reaction was monitored by liquid chromatography-mass spectrometry, showing the desired mass (a main peak with the desired mass was detected). 1.0 M hydrochloric acid solution (10.0 mL) was added to the reaction mixture until pH=6. The mixture was diluted with water (75.0 mL) and extracted with DCM (120 mL * 3). The mixture was freeze-dried to give target A001A (9.0 g, 5.96 mmol, 93.9% yield, >95% purity, HCl) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.96 (br t, J =5.4 Hz, 3 H), 7.67 (d, J = 8.9 Hz, 3 H), 7.51 (s, 1 H), 4.27 (d, J = 8.4 Hz,3 H), 3.75-3.80 (m, 6 H), 3.70 (br d, J = 10.0 Hz, 6 H), 3.49 (br d, J = 4.0Hz, 31 H), 3.37-3.42 (m, 12 H), 3.30 (br d, J = 6.1 Hz, 4 H), 3.20 (br d, J =5.8 Hz, 6 H), 2.99 (s, 2 H), 2.30 (br t, J = 6.4 Hz, 6 H), 1.80 (s, 9 H). Liquid chromatography-mass spectrometry (LC-MS) showed a retention time of 0.966 min, MS calculated value: 1396.6, measured value: [M + 2H] 2+ = 699.1.

[0807] Example 18

[0808] Chemical synthesis. Procedure for preparing TBT103.

[0809]

[0810] To a solution of target A001A (500 mg, 357 μmol, 1.00 equivalent) and N-hydroxysuccinimide ester of 3-maleimide propionate (142.869 mg, 536 μmol, 1.50 equivalent) in dimethylformamide (5 mL), N,N-diisopropylethylamine (46.2 mg, 357 μmol, 59.1 μL, 1.00 equivalent) was added, followed by degassing and purging three times with nitrogen. The mixture was stirred at 0 °C for five minutes under a nitrogen atmosphere. Liquid chromatography-mass spectrometry showed the desired mass (a main peak with the desired mass was detected). The reaction mixture was directly purified by preparative high-performance liquid chromatography (AcOH conditions) to give TBT103 as a colorless solid (251.2 mg, 156 μmol, 43.7% yield, 96.5% purity). Retention time by liquid chromatography-mass spectrometry = 1.382 minutes, MS calculated value: M av = 1548.59, [M + H] + = 1548.9, [M + 2H] 2+ = 775.1.

[0811]

[0812] Example 19

[0813] Membrane proteome array

[0814] Antibody deconvolution and specificity analysis The membrane proteome array (MPA) is a platform for analyzing the specificity of antibodies and other ligands targeting human membrane proteins. MPA is used to determine antibody target specificity and deconvolve orphan antibody / ligand targets. The MPA platform uses flow cytometry to directly detect antibodies binding to membrane proteins expressed in unfixed cells. Target proteins possess native conformations and appropriate post-translational modifications.

[0815] The reactivity of the ligands with our library of over 6,000 human membrane proteins was tested. This library includes 94% of all single-transmembrane, multi-transmembrane, and GPI-anchored proteins, including GPCRs, ion channels, and transporters. Identified targets were validated in a secondary screening to confirm reactivity.

[0816] Materials and Methods Screening on the membrane proteome array (MPA) was performed at Integral Molecular, Inc. The MPA is a protein library consisting of 6,000 different human membrane protein clones, each overexpressed in live cells via expression plasmids. Each clone was individually transfected into individual wells of a 384-well plate and incubated for 24 hours. See Tucker et al., Proc. Natl. Acad. Sci. USA. 115, E4990–E4999 (2018). Cells expressing each individual MPA protein clone were arranged in duplicate in a matrix for high-throughput screening. Prior to screening on the membrane proteome array, the concentrations of test ligands for screening were determined on cells expressing positive (tethering protein A) and negative (simulated transfection) binding controls, and then detected by flow cytometry using fluorescently labeled secondary antibodies. Each test ligand was added to the MPA at a predetermined concentration, and binding across the entire protein library was measured on an Intellicyt iQue using fluorescently labeled secondary antibodies. Each array plate contained positive (Fc binding) and negative (empty vector) controls to ensure inter-plate reproducibility. Using serially diluted test antibodies, interactions between the test ligands and any targets identified by the MPA screening were confirmed in a second flow cytometry experiment, and target identity was re-verified by sequencing.

[0817] The final selection results are summarized in the table below. This project was conducted in three phases:

[0818]

[0819]

[0820] Optimization of ligand concentrations for screening Serial dilutions of each test ligand were determined by incubation with known targets, and target binding was measured by flow cytometry. The optimal ligand concentration for screening was selected based on a combined assessment of binding strength and background signal (left) as well as the incidence of high background events.

[0821] Membrane proteome array screening To identify ligand-binding targets, approximately 6,000 different membrane proteins were expressed in the individual wells of HEK-293T cells arranged in a 384-well plate. The cells were then matrixed by aggregating the individual columns and rows of each plate, so that each protein was represented by a unique combination of two distinct wells in the matrix plate.

[0822] Targets were identified by detecting ligand binding to overlapping column and row pools (using previously optimized conditions), allowing for specific deconvolution. Each individual membrane protein target was assigned a value corresponding to its unique row and column pool binding value, and targets exhibiting binding signals >3 standard deviations above background in both wells were selected for downstream validation experiments. The resulting paired binding values ​​were then normalized and transformed to obtain a single numerical value for the binding of the tested ligand to each target protein. Non-specific fluorescence was defined as any value three standard deviations below the mean background value.

[0823] Membrane proteome array screening results The binding of each ligand to MPA at previously determined optimal concentrations was tested by flow cytometry. Binding interactions confirmed in downstream validation studies are shown in blue, and any proteins that failed validation were removed.

[0824] Target validation To validate any identified off-target interactions, cells were transfected in 384-well configurations using plasmids, protein A, or separate vectors encoding the identified targets. After incubation, four four-fold dilutions (starting from 20 µg / ml) of each test ligand were added to the transfected cells, and ligand binding was then detected using high-throughput immunofluorescence flow cytometry.

[0825] Determine the mean fluorescence intensity (MFI) for each ligand dilution. Only validated targets that exhibited a dose-response relationship and a mean fluorescence intensity ≥2 times higher than the background at two consecutive concentrations were tested.

[0826] Validation results of newly identified ligand targets The newly identified target was incubated with a series of ligand dilutions, and binding was measured by flow cytometry. A target was validated if it exhibited a binding signal ≥2-fold higher than the background at two consecutive test concentrations.

[0827] Example 20

[0828] Surface plasmon resonance. In vitro assessment to characterize mechanistic targets. Combine.

[0829] Surface plasmon resonance measurement Surface plasmon resonance (SPR) assays are a biophysical method that provides a label-free, real-time approach to measuring biomolecular interactions, including binding affinity, kinetics, and specificity. The assay is performed on a biosensor platform where ligands immobilized on a sensor chip bind to analytes flowing in solution. The resonance signal (RU) plotted based on analyte concentration is fitted to the K signal via a steady-state method. D .

[0830] The binding capacity of IgG4 binding sites can be measured using surface plasmon resonance (Biacore™ and IBIS-MX96 systems) and biolayer interferometry (ForteBio™ Octet™ system). This assay can be performed, for example, on a Biacore S200 instrument with high sensitivity and medium-to-low throughput, following the manufacturer's instructions. IgG4 binding acts as a ligand coupled to the surface of the sensor chip. As the analyte flows in a solution above the immobilized ligand, the binding to the sensor chip surface causes a change in refractive index proportional to the binding mass.

[0831] IgG4 Fc surface plasmon resonance scheme

[0832] background The purpose of this assay is to determine the binding of the bifunctional conjugate to IgG4 Fc using surface plasmon resonance.

[0833] This protocol was developed to confirm the target binding of protein-based degraders and pre-conjugated intermediates (monofunctional binders) to the IgG4 Fc protein. The experimental setup used a Biacore S200 instrument, but is compatible with other Biacore instruments such as the 8K or 8K+.

[0834] preparation According to the manufacturer's instructions, lyophilized IgG4 Fc protein was reconstituted in diH2O. For 200 µg package sizes of human IgG4 Fc protein from ACROBiosystems, the lyophilized material was reconstituted with 400 µL diH2O and gently mixed for 30 minutes at room temperature. The mg / mL reconstituted IgG4 Fc should be aliquoted to more than 10 µg of material per vial and stored at -80°C. The inventors avoided repeated freeze-thaw cycles.

[0835] Prior to the assay, IgG4 Fc was immobilized onto a CM5 chip using amine coupling, targeting 550 RU. hIgA1 was used as a reference surface.

[0836] The inventors equilibrated the CM5 sensor chip and sodium acetate buffer to room temperature by placing them on a workbench for about twenty minutes.

[0837] The inventors prepared a 1x HBS-P run buffer by mixing 100 mL of 10x HBS-P with 900 mL of ultrapure water. The contents were filtered through a 0.22 µm PES membrane (Corning #431174). Fresh run buffer should be prepared before each experiment.

[0838] Surface fixing Dilute IgG4 Fc protein and hIgA1 reference surface antibody to 20 µg / mL each in 10 mM sodium acetate buffer, pH 5.0. Prepare reagents from the amine conjugation kit using the volume and rack orientation indicated on the instrument protocol. Set the target fixation level to 550 RU.

[0839] Surface plasmon resonance binding determination (single-cycle kinetics)

[0840] (1) The instrument is equipped with a CM5 chip and is perfused three times with 1x HBS-P running buffer.

[0841] (2) Dilute the test molecule to the highest concentration of 1 µM in 1x HBS-P. The recommended minimum volume is 200 µL. Always use the same 1x HBS-P run buffer as the preparation solution perfused through the system to dilute the test molecule.

[0842] (3) In a 96-well plate, the highest concentration of the test sample is diluted 4-fold at six points. This dilution series can be achieved by aspirating 50 µL from the highest concentration dose and mixing it into 150 µL of 1x HBS-P, and so on until the last (lowest concentration) point.

[0843] (4) Add start-up or blank conditions to the appropriate wells according to the instrument program requirements (1x HBS-P buffer only).

[0844] (5) A 50% dimethyl sulfoxide regeneration solution was prepared by mixing equal volumes of dimethyl sulfoxide and 1x HBS-P.

[0845] (6) Allow the scheme to run and fit the affinity based on the single-cycle dynamic trajectory.

[0846]

[0847] Binding was confirmed by surface plasmon resonance.The inventors performed surface plasmon resonance assays to confirm the binding of the IgG4 Fc nanobody. Prior to the assay, the inventors immobilized the IgG4 Fc onto the CM5 sensor chip via amine coupling. The anti-IgG4 fragment was administered at doses ranging from 1 nM to 1 µM and characterized by single-cycle kinetics at a contact time of 60 seconds, a dissociation time of 60 seconds, and a flow rate of 30 µL / min.

[0848] The inventors obtained full-length and fragment domains of IgG4 labeled, for example, with biotin, to generate, for example, a 12 kDa recombinant antibody fragment with biotin conjugation. The inventors also labeled IgG4 with AF647 (3.5-6 dye molecules / protein) for use in surface plasmon resonance assays.

[0849] The inventors tested the unbonded material in surface plasmon resonance assays to ensure that chemical treatments did not reduce the binding of immunoglobulin regions.

[0850]

[0851] The results of surface plasmon resonance IgG4 degrader target binding assay confirmed the binding.

[0852] The inventors demonstrated that the commercial anti-IgG4 nanobody binds to human IgG4 Fc. In characterization with n=2, approximately 20 nM of K was obtained. D At concentrations up to 1 µM, no binding reactions were observed in the full-length IgG1-3 and IgG1-3 Fc regions.

[0853]

[0854] The steady-state affinity results obtained from the surface plasmon resonance determination are consistent with the obtained single-cycle kinetics.

[0855]

[0856] Using this surface plasmon resonance method, the inventors demonstrated the binding of the anti-IgG4 Fc nanobody ABT301 to IgG4 Fc. The anti-IgG4 Fc nanobody ABT301 exhibits a moderate nM binding affinity for IgG4 Fc.

[0857] Additional measurement information

[0858] preparation The inventors prepared the running buffer - 1X HBS-P by diluting 100 mL of 10x HBS-P in 900 mL of Milli-Q water. The buffer was filtered through a Corning 431174 0.22 µm PES membrane.

[0859] Sample preparation Prepare 1 µM conjugate samples in HBS-P (using the same freshly prepared buffer as the run buffer for the assay):

[0860] Prepare a 1000 µL sample at a concentration of 1 µM in 1X HBS-P buffer using the accompanying protocol (also noted above). Prepare 200 µL of AGN402 to conserve material.

[0861] (1) Use a multichannel pipette to add 150 µL of 1X HBS-P to the A1:F5 well.

[0862] (2) Add 200 µL of 1 µM sample to the appropriate well.

[0863] (3) Using a multichannel pipette, transfer 50 µL from the highest dilution series and mix in the next row to create a 1:4 series dilution with 6 points from right to left.

[0864] (4) Add 200 µL of 1X HBS-P to the G1 well as a buffer control.

[0865] (5) Mix 1 mL of DMSO with 1 mL of 1X HBS-P to prepare a 50% DMSO regeneration buffer. Add the regeneration buffer to a 4 mL plastic vial according to the above preparation and place it at position A1 on reagent rack 2.

[0866] In the Biacore S200 control software Before screening, perfuse the run buffer (1X HBS-P) three times by selecting "Multiple Perfusions" in the Tools menu. These selections are followed by template / wizard prompts to set up the protocol in the Biacore S200 software.

[0867]

[0868] Example X

[0869] Subtype selectivity

[0870] The anti-IgG4 Fc nanobody ABT301 did not respond to full-length IgG1, full-length IgG2, full-length IgG3, IgG1 Fc, IgG2 Fc or IgG3 Fc at concentrations up to 1 µM.

[0871]

[0872]

[0873] The inventors demonstrated almost no cross-species reactivity. Results showed no binding in non-human species, not even in rhesus monkeys. Sequence alignment revealed that IgG3 and IgG4 were similar at the binding site for cynomolgus monkey sequences.

[0874] Example 21

[0875] Determination of ternary complexes for pharmacological activity patterns based on characterization mechanisms .

[0876] The inventors performed a cell-based TR-FRET assay to evaluate ternary complexes on cell surfaces. The purpose of this assay was to screen IgG4 degradation conjugates in a previously developed FRET-based ternary complex formation assay that used AlexaFluor647-labeled anti-hIgG4 as the FRET receptor fluorophore.

[0877] The principle of TR-FRET is as follows: the donor fluorophore is excited by a laser or flash lamp and emits light. If close, the receptor fluorophore is excited by the donor's energy. The resulting signal emitted from the receptor is proportional to the level of interaction. See Li et al., J. Biomol. Screen., 20(7), 869-75 (August 2015) and Wen et al., AAPS J., 22(3), 68 (April 16, 2020). The TR-FRET assay is an in vitro assessment used to characterize the mechanism of pharmacological action. The TR-FRET assay measures the induced proximity of the bifunctional degrader moiety to verify the degrader-induced ASGPR-IgG4 proximity.

[0878] The inventors used AF647-labeled anti-hIgG4 secondary antibody as the receptor fluorophore in the TR-FRET assay. The assay conditions were: 18 µL of reaction solution containing 6.3 nM hIgG4 (Abcam), 25 nM ASGPR1, 2.5 nM streptavidin-europene cavitary compound, 20 nM AF-647-labeled anti-hIgG4, and 0.1% dimethyl sulfoxide.

[0879] Measurement This assay was developed to confirm the formation of a trimeric ternary complex between ASGPR1 and IgG4 induced by a heterobifunctional degrader. An Echo acoustic liquid processor was used to dispense the dimethyl sulfoxide negative control and the AGN406 positive control, while a handheld multichannel pipette was used to dispense the aqueous dilution of the test molecules.

[0880] Measurement preparation The inventors slowly thawed rASGPR on ice from storage conditions of -80°C.

[0881] The inventors prepared AGN406 at a concentration of 12.5 μM (100x final maximum concentration) in 100% dimethyl sulfoxide. The inventors added AGN406 and dimethyl sulfoxide to Echo-qualified microplates for allocating positive and negative control assays.

[0882] The inventors vertically doubled AGN406 in 384-well v-plates (sixteen spots in total) by mixing 25 µL in 25 µL of 1x buffer, starting from the highest concentration of 250 μM in Echo qualified source plates.

[0883] Measurement steps (1) The inventors prepared an appropriate volume of 1x assay buffer: 25 mM HEPES pH 7.5, 150 mM sodium chloride, 5 mM calcium chloride, 1 mM dithiothreitol, and 0.01% TWEEN-20.

[0884]

[0885] (2) The inventors prepared 50 μL of the degradation agent test sample at a concentration of 30 μM in 1x assay buffer as the highest dose in the dilution series.

[0886] (3) Using the Echo liquid processor, the inventors dispensed 180 nL of dimethyl sulfoxide (negative control), 180 nL of 12.5 μM AGN406 (positive assay control), and 180 nL of AGN406 dose control into the appropriate wells of a 384-well OptiPlate. These wells were then backfilled with 6 μL of 1x assay buffer for volume scaling.

[0887] (4) The inventors used an electronic multichannel repeatable pipette to add 6 μL of the degradation agent dilution to the appropriate well of the test plate. The plate was then sealed and set aside.

[0888] (5) The inventors prepared a 3x protein mixture containing 75 nM ASGPR1 and 18.75 nM IgG4 in a 1x assay buffer.

[0889]

[0890] (6) Using a Multidrop liquid processor, the inventors dispensed 6 μL of protein mixture into all wells of an assay plate containing a degradation agent diluent and a control.

[0891] (7) The inventors sealed the plate, centrifuged it at 1000 RPM for one minute, and incubated it at room temperature for sixty minutes.

[0892] (8) The inventors prepared a 3x detection mixture in a 1x assay buffer: 7.5 nM streptavidin-europene cavitation compound and 60 nM mouse anti-human IgG4 Fc-AlexaFluor647.

[0893]

[0894] (9) Using the Multidrop Combi liquid processor, the inventors dispensed 6 μL of the test mixture into all wells of the test plate.

[0895] (10) The inventors sealed the plate, centrifuged it at 1000 RPM for one minute, and incubated it at room temperature for sixty minutes.

[0896] (11) The inventors recorded FRET using excitation at 320 nm and emission at 665 nm and 615 nm on an Envision multi-label reader. Data were output as .csv files, with FRET ratio data reduction performed on the reader. TR-FRET values ​​were calculated as [(665 / 615)*1000] and plotted as the response at each test concentration of the degradant. The measured signal-to-background ratio was calculated as the average signal of the fixed-concentration positive control (AGN406) divided by the average signal of the negative control (dimethyl sulfoxide) wells. s / b and Z' were calculated. Dose-response data were formatted to plot two replicates side-by-side.

[0897]

[0898] Reagents for protein biotinylation for purification, immobilization, or sensitive protein detection are commercially available from AviTag™ Technologies (GeneCopoeia, Rockville MD, USA).

[0899] The inventors demonstrated the in vitro activity of the degrader-induced ASGPR-IgG4 proximity, as measured by the formation of a ternary complex via TR-FRET assay. The induced proximity between ASGPR and IgG4 generates a specific energy transfer signal. The TR-FRET signal depends on the MoDE-induced proximity of ASGPR and IgG4. The TR-FRET signaling of the VHH-based degrader molecule validates the bifunctional mechanism of action.

[0900]

[0901] The inventors titrated AGT406 in triplicate at sixteen points, with concentrations ranging from 10 µM to 0.3 nM.

[0902] The inventors used an assay based on HEK293 fluorescent cells to assess the formation of ternary complexes. This HEK293 fluorescent cell-based assay allows those skilled in the biomedical field to monitor the kinetics of ternary complex formation in cells.

[0903] The inventors have also used other assays to assess event-driven mechanisms of action. For example, see Pettersson & Crews, Drug Discovery Today Technologies, 31, 15-27 (Elsevier BV, 2019). This assay allows a person of ordinary skill in the biomedical field to monitor the kinetics of ternary complex formation in cells.

[0904] The inventors also used NanoBRET, a bioluminescent resonance energy transfer (BRET) assay that measures protein-protein interactions (PPIs) in living cells. NanoBRET uses a small, bright luciferase called Nanoluc to label intracellular proteins and a long-wavelength fluorophore called HaloTag as an energy acceptor. When two proteins are very close, energy is transferred from Nanoluc to HaloTag, resulting in fluorescence emission at a specific wavelength. NanoBRET can be used to analyze binding events, signal transduction pathways, and acceptor transport.

[0905] Example 23

[0906] Assay for endocytosis of IgG4 compounds

[0907] The inventors used IgG4 from Sydlabs (recombinant and conjugated with the fluorophore IF488).

[0908] For routine culture of HEK-ASGPR1 cells, the inventors used DMEM high glucose containing 10% fetal bovine serum, 2 mM glutamine and 1% P / S + 200 ug / ml genimycin.

[0909]

[0910]

[0911] The inventors rinsed the cells once with Dulbecco phosphate-buffered saline, which is free of calcium and magnesium.

[0912] The inventors then harvested HEK-ASGPR1 cells as follows: 5 ml of accutase was incubated at 37°C in a T175 flask for five minutes. 10 ml of culture medium was added and pipetted. The mixture was then centrifuged at 1000 rpm for five minutes.

[0913] Based on the plate diagram, the inventors used DMEM high glucose (31053028) without phenol red + 10% fetal bovine serum + 2 mM glutamine + 1% P / S + 200 μg / ml genimycin, and inoculated 30k / well in a 96-well plate, 100 µl per well.

[0914] The inventors used a poly-D-lysine 96-well plate (Corning Incorporated #3842).

[0915] Cells were grown overnight in an incubator. The next day, the inventors added 50 nM IgG4 to appropriate culture medium (Opti-MEM + 200 μg / ml G418 and the compound). The inventors performed an eight-point dose response at the highest concentration of 500 nM and a two-fold dilution. For the last point, the dilution was ten times the previous concentration.

[0916] The inventors incubated the assay overnight using an Incucyte instrument and took readings at 0, 4, 6, 10, 12, 14, 16, 20 and 24 hours.

[0917] At 24 hours, the inventors rinsed with calcium / magnesium-free Dulbecco phosphate-buffered saline to reduce background. They then added 100 μl / well of culture medium (Opti-MEM + 200 μg / ml genimycin) and scanned the gain using Incucyte.

[0918] Example 24

[0919] In vitro degradation assay .

[0920] This mechanistic degradation assay was performed via direct, low-throughput Western blot measurement. Western blot assays can be used to measure degradation in HEK293-ASGPR1 cells.

[0921] Measurement Workflow

[0922] Day 0 The inventor used 0.5 x 10 6 Cells were seeded per well into a 6-well plate.

[0923] Day 1 100 nM IgG4-AF488 + 60 nM compound were treated overnight.

[0924] Day 2 The inventors performed three washes with DPBS. Cells were lysed at T0 hour (maximum) and other time points. The inventors added bafloxacin (a lysosomal inhibitor) to some wells.

[0925] Day 3 It ruptures at a point in time, within 24 hours.

[0926]

[0927] AGT402 can cause almost complete degradation of IgG4 within five hours after washing. Bafloxacin inhibits the degradation of IgG4 (the degradation is lysosome-dependent). Caianiello et al., Nature Chemical Biology (2021) described an ASGPR-dependent mechanism of degradation induced by extracellular target molecular degraders (MoDEs). Bafloxacin inhibits the degradation of AF-488-conjugated IgG4 because the degradation is lysosome-dependent.

[0928] Example 25

[0929] LysoLgBiT assay

[0930] The LysoLgBiT assay is a tool for real-time study of the internalization and degradation of target proteins (POIs). By combining lysosome-specific signaling with protein labeling strategies, this assay offers both precision and adaptability, making it suitable for a wide range of applications. LgBiT expression vectors and stable cell lines are commercially available from Promega (catalog number N2681). See U.S. Patent No. 9,797,889 (Dixon et al.).

[0931] The characteristics of LysoLgBiT assay include: :

[0932] LAMP1-LgBiT fusion protein is a lysosome-specific fusion protein that provides robust and accurate readings for the localization and degradation of HiBiT-tagged drugs.

[0933] The HiBiT tag is an 11-amino acid peptide that can be attached to reagents using a simple and efficient copper-free click chemistry method. This method minimizes interference with reagent function and simplifies assay preparation. Fluorescent protease substrates achieve specificity through the integration of customizable linker sequences.

[0934] The fusion of ASGPR1 HiBiT / NanoLuc proteins in HEK293 cells enables real-time monitoring of protein internalization kinetics. Reduced luminescence is a direct indicator of internalization and subsequent lysosomal degradation.

[0935] Fluorescein / quencher pairs, such as DyLight 650 and IRDye QC-1, allow for the assessment of protein degradation, as described in established protocols. See Li et al., A cell-based internalization and degradation assay with an activatable fluorescence–quencher probe as a tool for functional antibody screening. Journal of Biomolecular Screening, 20(7), 869-75 (August 2015).

[0936] Incucyte assays offer a streamlined workflow and cost-effective protein labeling, optimized for high-throughput imaging. Incucyte FabFluor is a pH-sensitive dye, a Fab fragment conjugated to a pH-dependent fluorophore, which binds to Fc. This dye is optimized for Incucyte optics and analysis software.

[0937] Flow cytometry achieves high sensitivity and reduces background noise by gating labeled cell populations.

[0938] By attaching a HiBiT tag, LysoLgBiT assays can be applied to any reagent. The HEK293 HiBiT / NanoLuc fusion typically does not require reagent-specific modification and has the potential for flux expansion and broad real-time applicability.

[0939] Example 26

[0940] In vitro toxicology studies

[0941] In off-target assays conducted by the inventors, an in vitro toxicology safety panel was used to measure the cytotoxicity of the drug. The inventors used HEPG2 cells for toxicological evaluation, for example, in the CellTiter-Glo assay.

[0942] Furthermore, metabolite identification (MetID) is a crucial and indispensable part of drug discovery and development. In this context, metabolism describes the breakdown of exogenous substances and their conversion into molecules that can be excreted from the body. The inventors evaluated MetID in mice both in vitro and in vivo (blood, urine, and liver).

[0943] These results are sufficient to continue drug development.

[0944] The inventors also conducted cell-based screening assays to measure cytokine release.

[0945] Example 27

[0946] In vivo toxicology studies

[0947] The inventors are conducting toxicological studies to measure intravenous compatibility.

[0948] Selection of toxicological species The inventors are conducting toxicological assays on several mammalian model species.

[0949] Rats can be selected as the second toxicological species. The inventors have experience in conducting rat toxicology using intravenous bolus tests and measuring cytokine responses.

[0950] Humanized IgG4 miniature pigs can be selected as a second toxicological species. The inventors can evaluate the pharmacokinetics of live miniature pigs and collect miniature pig plasma to confirm binding with IgG4.

[0951] Because monkeys have an immune system similar to humans, they can be chosen as the second toxicological species. Rabbits only have one IgG isotype, so they are not of high value.

[0952] Example 28

[0953] In vivo tolerance test

[0954] The inventors conducted tolerance tests, starting with in vivo mouse assays (100 mg / kg) and then with in vivo rat assays (up to 1000 mg / kg).

[0955]

[0956]

[0957] Example 29

[0958] Physical stability

[0959] In readily available biological agents (20 mM histidine, pH 6.0, 8% sucrose), the VHH-Fc fusion complex showed no stability issues at a concentration of 90 mg / mL.

[0960] The inventors also confirmed this in plasma stability assays.

[0961]

[0962]

[0963] Example 30

[0964] Other in vivo assays using mouse models .

[0965] Efficacy determination of pemphigus vulgaris model Subcutaneous injection in BVMAB786 mice. Day 1: Administration of anti-Dsg3 IgG4AutoAb, followed shortly by administration of an IgG4 degrading agent. Day 2: Administration of ETA. Symptoms appeared 18 to 72 hours later. ETA should have no effect in the presence of the degrading agent. In the absence of the degrading agent, ETA should induce pemphigus vulgaris symptoms.

[0966]

[0967]

[0968] Anti-drug antibody (ADA) testing This was conducted in a mouse model. Many antibody drugs use IgG4 as their antibody.

[0969] Other assays using mice Validating the in vivo biological activity of the mechanism of action (pharmacokinetics / pharmacodynamics in ASGPR knockout mice). Pharmacokinetic and pharmacodynamic parameters were obtained from intravenous and subcutaneous mouse studies.

[0970] Example 31

[0971] In vivo consumption measurement

[0972] In vivo characterization validated the depletion of IgG4 by the VHH-ASGPR ligand bifunctional degrader (non-Fc fusion variant). The non-Fc fusion variant degrader also showed IgG4 depletion.

[0973]

[0974]

[0975]

[0976]

[0977]

[0978]

[0979]

[0980] Example 32

[0981] In vivo bioavailability assay

[0982] The inventors conducted a series of assays in mice and rats to gain insights into subcutaneous and intravenous administration routes using repeated dosing regimens to demonstrate the bioavailability of the administered drug in subjects.

[0983] Subcutaneous bioavailability The AGT402 Fc fusion bifunctional degrader demonstrated approximately 50% bioavailability in a mouse pharmacokinetic study.

[0984] Similarly, subcutaneous administration in rats has been used to measure the bioavailability of Fc and non-Fc drugs, revealing potential correlations and differences in cross-species pharmacokinetics.

[0985] Intravenous and subcutaneous administration Comparative studies have demonstrated the differences in bioavailability between intravenous and subcutaneous administration.

[0986] Data from the four agents, including an analysis of rebound effects, demonstrate key indicators for understanding dose-dependent behavior.

[0987] Ongoing research is refining bioavailability data and collecting additional time points beyond 24 and 48 hours to provide a comprehensive understanding of long-term systemic circulation.

[0988] Example 33

[0989] Analytical size exclusion chromatography determination

[0990] The inventors used analytical size exclusion chromatography (SEC) to characterize and quantify the size distribution of the drug substance within a sample of active pharmaceutical ingredient. This assay detects drug aggregates and fragments. This information is used for quality control during development and manufacturing processes. All currently available analytical size exclusion chromatography results appear promising. Developability data revealed that for the VHH-Cys molecule, size exclusion chromatographic purity decreased only slightly (<95%) under thermal and pH stress conditions, showing a shift towards acidic peaks as observed by imaging capillary isoelectric focusing. For the VHH-Fc fusion molecule, the effect was less pronounced and limited to thermal stress conditions. The data suggest that the Fc fusion molecule imparts a stability advantage.

[0991] Example 34

[0992] Human subjects selected for drug administration

[0993] Outpatient studies can be conducted on MuSK patients with myasthenia gravis. Subcutaneous application, designed for home use, improves patient convenience and adherence. The quantitative myasthenia gravis score provides a reliable indicator for assessing disease severity and treatment outcomes. Disease severity in MuSK myasthenia gravis is more closely related to IgG4 autoantibody levels. Compared to classic myasthenia gravis, MuSK myasthenia gravis demonstrates a stronger clinical response.

[0994] MuSK-specific IgG4 autoantibodies are robust biomarkers that provide a precise method for identifying eligible patients. This precision can aid in clinical trial recruitment.

[0995]

[0996] Example 35

[0997] Human dosage determination

[0998] The inventors are developing a chemometric method for human dose determination. The inventors used the following premises for initial human dose determination: (1) IgG4 level: 40 mg / dL; (2) IgG4 synthesis rate: 23 mg / day; VHH: approximately 15 kDa; (3) VHH-Fc: approximately 80 kDa; (4) maximum drug concentration: 100 mg / mL subcutaneously at 50% BA; and (5) maximum drug concentration: 100% BA intravenously. The inventors refined the human pharmacokinetic and pharmacodynamic parameters based on intravenous and subcutaneous pharmacokinetic / pharmacodynamic measurements from ongoing assays in rats. The inventors further refined the human pharmacokinetic and pharmacodynamic parameters by comparing human dose parameters with literature data. The inventors are validating the method by estimating ASGPR levels and pharmacokinetic parameters.

[0999] Example 36

[1000] Drug efficacy

[1001] A person skilled in the biomedical field can use several standards known to them to evaluate the effectiveness of a drug.

[1002] The effectiveness of the medication can be determined using the Ray Auditory-Verbal Learning Test (RAVLT).

[1003] The effectiveness of the medication can be determined using the Montreal Holistic Cognitive Assessment (MOCA).

[1004] The effectiveness of the medication can be determined by the time it takes for the seizures to disappear or status epilepticus to cease without the use of rescue therapy.

[1005] Equivalent solution

[1006] Those skilled in the biomedical field will recognize or be able to determine numerous equivalents of the specific procedures described herein using experiments not exceeding those of conventional methods. Such equivalents are within the scope of this invention and are covered by the following claims. For example, pharmaceutically acceptable salts other than those specifically disclosed in the descriptions and examples herein may be used. Furthermore, it is an aim that specific items in the list of items or subsets of larger groups of items can be combined with other specific items, subsets of items, or larger groups of items, regardless of whether there is a specific disclosure in this specification to identify such combinations. Those skilled in the art will further recognize that the concept of this invention covers the hydrated open-chain form of the claimed compounds containing a succinimide moiety, produced by the ring-opening reaction (i.e., hydrolysis) of the succinimide moiety with water. Those skilled in the art can readily determine such ring-opening structures and their alternative representations. See, for example, Kai Zheng et al., Characterization of Ring-Opening Reaction of Succinimide Linkers in ADCs. Pharmaceutical Biotechnology 108 (1), 133-141 (January 2019). Scheme 1.

[1007] Some embodiments of the present invention can be practiced according to the following numbered paragraphs:

[1008] 1. A composition comprising: an IgG4 binding portion; a cell receptor binding portion, the cell receptor binding portion being capable of binding to hepatocytes or other degrading cells via a desialylate glycoprotein receptor (ASGPR) on hepatocytes or other cell receptors on the surface of degrading cells; and a linker portion (optionally a single peptide bond) connecting the IgG4 binding portion and the cell receptor binding portion.

[1009] 2. The material composition according to Example 1, wherein the IgG4 binding portion is a disclosed VHH affinity ligand variant antibody, a VHH affinity ligand variant, or an antigen-binding fragment thereof.

[1010] 3. The material composition according to Example 1, wherein the IgG4 binding portion is a polypeptide having a complementarity-determining region (CDR) for an anti-IgG4 VHH affinity ligand variant.

[1011] 4. The material composition according to Example 1, wherein the IgG4 binding portion comprises three regions having structures of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 according to the Chothia numbering scheme.

[1012] 5. The material composition according to Example 1 has the following structure: R CN −(Xaa)y−R CC , [AGN101]、 [AGN102] [AGN103] or [AGN104] or its salt.

[1013] 6. The composition of the substance according to Example 1, wherein the pharmaceutical agent has the formula... The structure of [AGN102] or a pharmaceutically acceptable salt thereof, wherein: each of a and b is independently an integer of 1 or greater; each AT is an IgG4 binding moiety or a fragment thereof; L is a linker moiety; and each TBT is independently a cell receptor binding moiety that binds to hepatocytes or other degrading cells via the desialylate glycoprotein receptor (ASGPR) of hepatocytes of the patient or subject or other cell receptors on the surface degrading cells, wherein the IgG4 binding moiety is an IgG4 VHH affinity ligand variant or an antigen-binding fragment thereof.

[1014] 7. The composition of substances according to Example 1, wherein the pharmaceutical agent has the formula... [AGN106] or its salts thereof, wherein the composition of the substance has additional elements described in this specification.

[1015] 8. The composition of the substance according to Example 1, wherein the cell receptor binding portion has the following structure: or ;where R A It is a C1-C3 alkyl group, which is optionally substituted with one to five halogenated groups, preferably fluorinated groups, and preferably R A It is a methyl or ethyl group, which is optionally substituted by one to three fluorinated groups; Z A It is -(CH2) IM -O-(C...

Claims

1. A composition comprising: IgG4 binding site, The cell receptor binding portion, which can bind to hepatocytes or other degrading cells via the desialylate glycoprotein receptor (ASGPR) on hepatocytes or other cell receptors on the surface of degrading cells, and The connector portion connects the IgG4 binding portion and the cell receptor binding portion.

2. The composition according to claim 1, having the following structure: R CN −(No)y−R CC , [AGN101], [AGN102], [AGN103], or [AGN104] Or its salt.

3. The material composition according to claim 1, wherein the material composition is selected from the group consisting of: AGN401, AGN402, AGN403, AGN404, AGN501, AGN502, AGN508 and AGN509.

4. The composition according to claim 1, wherein the IgG4 binding portion is an anti-IgG4 antibody or an antigen-binding fragment thereof.

5. The composition according to claim 1, wherein the IgG4 binding portion is a VHH affinity ligand variant antibody, a VHH affinity ligand variant, or an antigen-binding fragment thereof.

6. The composition of claim 1, wherein the IgG4 binding portion comprises three regions having structures of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

4.

7. The composition of claim 1, wherein the IgG4 binding portion is selected from the group consisting of: ABT301, ABT304, ABT305, ABT306, ABT310, ABT311, ABT312, ABT313, ABT501, ABT502, ABT503 and ABT504.

8. The composition according to claim 1, wherein the cell receptor binding portion has the following structure: or ; Where R A It is a C1-C3 alkyl group, which is optionally substituted with one to five halogenated groups, preferably fluorinated groups, and preferably R A It is a methyl or ethyl group, which is optionally substituted by one to three fluorinated groups; Z A It is -(CH2) IM -O-(CH2) IM S-(CH2) IM NR M -(CH2) IM ;C(O)-(CH2) IM -; containing 1 to 8, preferably 1 to 4, ethylene glycol residues of a PEG group; or -C(O)(CH2) IM NR M The group (preferably containing a group comprising 1 to 8 ethylene glycol residues, preferably 2 to 4 ethylene glycol residues) and Z B It does not exist; it is (CH2). IM C(O)-(CH2) IM -or C(O)-(CH2) IM -NR M .

9. The composition according to claim 1, wherein the cell receptor binding portion is selected from the group consisting of TBT103, TBT104, TBT105 and TBT106.

10. The composition of claim 1, used as a medicine.

11. The composition of claim 1, used for removing IgG4 from a subject.

12. The composition of claim 1, used to treat a subject or patient with a disease state or symptom associated with IgG4 upregulation.

13. The composition of claim 1, used for treating IgG4-related diseases in a subject or patient.

14. A method for preparing a composition according to claims 1 to 9, wherein the method comprises a conjugation step of linking an IgG4 binding portion to a cell receptor binding portion via a linker portion, wherein the conjugation step is selected from the group consisting of a MATE conjugation step, a maleimide conjugation step, and a sorting enzyme conjugation step.

15. A pharmaceutical composition comprising the material composition according to claims 1 to 9 and a pharmaceutically acceptable excipient.

16. A method for removing IgG4 from a subject or patient, the method comprising administering to the subject or patient a composition of substance according to any one of claims 1 to 9.

17. A method of treating a subject or patient with a disease state or symptom associated with IgG4 upregulation, the method comprising administering to the subject or patient an effective amount of the composition of the substance according to any one of claims 1 to 9.

18. A method of treating a subject or patient with IgG4-related disease, the method comprising administering to the subject or patient an effective amount of the composition of the substance according to any one of claims 1 to 9.

19. A composition comprising: A first material composition, the first material composition comprising: IgG4 binding site, The cell receptor binding portion, which can bind to hepatocytes or other degrading cells via the desialylate glycoprotein receptor (ASGPR) on hepatocytes or other cell receptors on the surface of degrading cells, and A connector portion, wherein the connector portion connects the IgG4 binding portion and the cell receptor binding portion; and At least one other material composition, said at least one other material composition comprising the following portions: The cell receptor binding portion, which can bind to hepatocytes or other degrading cells via the desialylate glycoprotein receptor (ASGPR) on hepatocytes or other cell receptors on the surface of degrading cells, and The connector portion connects the IgG4 binding portion and the cell receptor binding portion.

20. The composition according to claim 19, wherein the additional agent has a structure selected from the group consisting of: formula LG−RG−L RM (−TBT) b LG−RG−H and their combinations.

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