WO2025158412A1 - BIFUNCTIONAL DEGRADERS OF IgG4 IMMUNOGLOBULINS - Google Patents

Abstract

A composition of matter including an IgG4-binding moiety, a cellular receptor-binding moiety which binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes or other degrading cells in a patient or subject, and optionally, a linker moiety connecting the IgG4-binding moiety and the cellular receptor-binding moiety, wherein the composition of matter is useful for removing IgG4 from a subject or patient.

Description

BIFUNCTIONAL DEGRADERS OF IgG4 IMMUNOGLOBULINS FIELD OF THE INVENTION [001] The invention generally relates to medicinal preparations characterized by the non-active ingredients used, e.g., carriers or inert additives, targeting or modifying agents chemically bound to the active ingredient, the non-active ingredient being chemically bound to the active ingredient, e.g., polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g., an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant, and particularly to bifunctional molecules which have a circulating protein-binding moiety linked through a linker group to a cellular receptor- binding moiety for the treatment of an IgG4-related disease. BACKGROUND OF THE INVENTION [002] IgG4-related disease (IgG4-RD), formerly known as IgG4-related systemic disease, is a chronic inflammatory condition characterized by tissue infiltration with lymphocytes and IgG4-secreting plasma cells. In most people with this disease, serum IgG4 concentrations are elevated during an acute phase. Kamisawa et al., The Lancet, 385 (9976), 1460–1471 (April 11, 2015). [003] An international panel of experts 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 treatment is required for all cases of symptomatic, active IgG4-related disease. Treatment is advised for some organ manifestations, such as aortitis, retroperitoneal fibrosis, proximal biliary strictures, tubulointerstitial nephritis, pachymeningitis, pancreatic enlargement and pericarditis. Some cases with asymptomatic IgG4-related disease also require treatment because some organs tend to not cause symptoms until the late stages of disease. [004] The recommended first-line agent for induction of remission is glucocorticoids unless contraindications exist, but this treatment does not address the disease cause. The goal of glucocorticoid treatment is the induction and maintenance of remission to prevent progression of fibrosis and organ destruction in affected organs. Recurrences during or after tapering of glucocorticoids are frequent. Thus, there remains a need in the biomedical art for new medicines capable of treating or slowing down progression of IgG4-related disease.

1 30123-WO-PCT SUMMARY OF THE INVENTION [005] The invention is directed to bifunctional compositions of matter (agents) capable of binding, removing from blood circulation, and degrading IgG4 immunoglobulins. [006] In one embodiment, the invention provides a composition of matter (an agent) comprising: an IgG4-antibody-binding moiety, a cellular receptor-binding moiety that can bind to hepatocytes or other degrading cells through the asialoglycoprotein receptors (ASGPR) of hepatocytes or receptors on the surface of degrading cells in a patient or subject, and a linker moiety connecting the IgG4-binding moiety and the cellular receptor-binding moiety, wherein the linker moiety can be a peptide bond or a larger linker moiety. [007] In some embodiments, the composition of matter (agent) has the structure: [CRBM] is a cellular receptor-binding moiety; each [CON] is an optional connector chemical moiety which, when present, connects directly to [CPBM] or to [CRBM] or connects the [LINKER] to [CPBM] or to [CRBM], and [LINKER] is a chemical moiety that covalently attaches to one or more [CRBM] or [CPBM] groups, or a pharmaceutically acceptable salt, stereoisomer, solvate, or polymorph thereof. [008] In some embodiments, the composition of matter (agent) has the structure of: R^CN−(Xaa)y−R^CC,

2 30123- eof. [009] In some specific and nonlimiting embodiments, the composition of matter is selected from the group in TABLE 1. TABLE 1 ID NO. IgG4-binding moiety Cellular receptor binding Linkage moiety AGN401 VHH w/o hinge region; ABT306 βGN3, TBT103 thiosuccinimide AGN402 VHH-Fc fusion, ABT310 βGN3 via MATE, TBT105 MATE conjugation AGN403 VHH with hinge region βGN3, TBT106 sortase AGN404 VHH w/o hinge region βGN3, TBT106 sortase AGN501 VHH-G₄S-Cys, ABT502 βGN3, TBT103 thiosuccinimide AGN502 VHH-G₃ECS-Cys, ABT508 βGN3, TBT103 thiosuccinimide AGN508 VHH-G4S-his6-G4S-Cys ABT501 βGN3, TBT103 thiosuccinimide AGN509 ABT504 βGN3, TBT103 thiosuccinimide [0010] AGN402 has a lysine-based conjugation. In the IgG4-binding moiety, two same single chains are covalently linked by disulfide bonds (ABT310, Fc-fusion). To make the composition of matter, TBT105 is conjugated to lysine amines in the nanobody fusion. During the synthesis of AGN402, the leaving group is eliminated when the payload is conjugated to amines. [0011] AGN508 has a cysteine-based conjugation. The IgG4-binding moiety has one chain (ABT501, nanobody). To make the composition of matter, the maleimide-containing TBT103 is conjugated to C- terminal cysteine of ABT501 via a Michael-addition reaction. [0012] AGN501 has a cysteine-based conjugation.. The IgG4-binding moiety has one chain (ABT502, nanobody). To make the composition of matter, the maleimide-containing TBT103 is conjugated to C- terminal cysteine of ABT502 via a Michael-addition reaction. [0013] AGN502 has a cysteine-based conjugation. The IgG4-binding moiety has one chain (ABT503, nanobody). To make the composition of matter, the maleimide-containing TBT103 is conjugated to C- terminal cysteine of ABT503 via a Michael-addition reaction.

3 30123-WO-PCT [0014] AGN509 has a cysteine-based conjugation. The IgG4-binding moiety has one chain (ABT504, nanobody). To make the composition of matter, the maleimide-containing TBT103 is conjugated to C- terminal cysteine of ABT504 via a Michael-addition reaction. [0015] AGN401 has a cysteine-based conjugation. The IgG4-binding moiety has one chain (ABT306, nanobody). To make the composition of matter, the maleimide-containing TBT103 is conjugated to C- terminal cysteine of ABT306 via a Michael-addition reaction. [0016] AGN403 has a sortase-ligation based conjugation. The IgG4-binding moiety has one chain (ABT304). To make the composition of matter, the pentaglycine-modified TBT106 is conjugated to a C- terminal pentapeptide motif LPETG of ABT304, via a sortase-catalyzed ligation. [0017] AGN404 has a sortase-ligation based conjugation. The IgG4-binding moiety has one chain (ABT305). To make the composition of matter, the pentaglycine-modified TBT106 is conjugated to C- terminal pentapeptide motif LPETG of ABT305, via a sortase-catalyzed ligation. [0018] In another embodiment, the IgG4-antibody-binding moiety is an antibody and antibody- derived binding protein. In a particular and nonlimiting embodiment, the complementary determining regions of the IgG4-antibody-binding moiety include the regions according to the Chothia numbering scheme having the structures of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. [0019] In another embodiment, the IgG4-antibody-binding moiety is a VHH nanobody. [0020] In some specific and nonlimiting embodiments, the IgG4 binding moiety is selected from the group in TABLE 2. TABLE 2 ID NO. SEQ ID NO. Protein structure ABT301 SEQ ID NO: 1 VHH nanobody (122 AAs) ABT304 SEQ ID NO: 9 VHH nanobody (122 AAs), G4S linker, C-term Sortase site, His6 tag ABT305 SEQ ID NO: 10 VHH nanobody with truncation of hinge, G4S linker, C-term Sortase site, His6 tag ABT306 SEQ ID NO: 11 VHH nanobody with truncation of hinge, G4S linker, His6 tag, rigid linker-Cys ABT310 SEQ ID NO: 12 VHH nanobody with truncation of hinge, G4S linker, hIgG1 hinge (C220A)-human IgG1 Fc-L253A/L254A/P329A ABT311 SEQ ID NO: 32 VHH nanobody (122 AAs), G4S linker, His6 tag ABT312 SEQ ID NO: 33 VHH nanobody with truncation of hinge, G4S linker, His6 tag ABT313 SEQ ID NO: 34 VHH nanobody with truncation of hinge, G4S linker, His6 tag, G2S linker, AviTag ABT501 SEQ ID NO: 59 VHH nanobody-G4S-his6-G4S-Cys ABT502 SEQ ID NO: 64 VHH nanobody-G4S-Cys ABT503 SEQ ID NO: 63 VHH nanobody-G3ECS-Cys ABT504 SEQ ID NO: 65 VHH nanobody-G4S-his6-Cys

4 30123-WO-PCT [0021] In another embodiment, the cellular receptor-binding moiety comprises an ASGPR-binding group according to the chemical structure: wherein the cellular receptor-binding moiety has additional elements described in this specification. [0022] In some specific and nonlimiting embodiments, the cellular receptor-binding moiety is selected from the group in TABLE 3. TABLE 3 ID NO. Chemical Structure TBT103

5 30123-WO-PCT TABLE 3 ID NO. Chemical Structure TBT104 NH O _OH O OH O O O_{O HN} OH H_{O O} N H O O HO O O O O N H HO O O H O O N N NH H H N O O_N^N O H N N HO O N O H H O O N N O O^O O O H H^{H N} N H O O O O H_O HN TBT105 TBT106 [0023] In another embodiment, the invention provides the composition of matter (agent) for use as a medicine. [0024] In another embodiment, the invention provides the composition of matter (agent) for use in treating or preventing a disease state or condition associated with the upregulation of IgG4 in a patient or subject. [0025] In one aspect, the administered composition of matter binds to an IgG4 antibody, especially although not exclusively an IgG4 autoantibody, in the circulation of a subject or patient to form an agent-antibody complex. After this agent-antibody complex is endocytosed by a cell, especially although

6 30123-WO-PCT not exclusively by a hepatocyte, it is released from the ASGPR through depletion of calcium from the endosome and changes in binding site amino acid protonation changes due to a decrease in pH. Endocytosed proteins are trafficked to endosomes, which then fuse with lysosomes. Lysosomal proteases then degrade endocytosed proteins, permanently removing them from circulation. [0026] In another embodiment, the invention provides a pharmaceutical composition including the composition of matter and at least one pharmaceutically acceptable excipient. [0027] In another embodiment, the invention provides a method of making the composition of matter wherein the method comprises a conjugation step, wherein the conjugation step results in an IgG4-binding moiety being linked to a cellular receptor-binding moiety. In another embodiment, the method of making the composition of matter comprises a conjugation step selected from the Markush group comprising a MATE conjugation step, a maleimide conjugation step, and a sortase conjugation step. [0028] In another embodiment, the invention provides a method of making a molecule wherein the IgG4-binding moiety is linked to another molecule or a device, wherein the method comprises a conjugation step selected from the Markush group comprising a MATE conjugation step, a maleimide conjugation step, a sortase conjugation step, and an AviTag™ conjugation step. [0029] In another embodiment, the invention provides a method of removing IgG4 in a patient or subject by administering an effective amount of the agent to the patient or subject. [0030] In another embodiment, the invention provides a method of treating or preventing a disease state or condition associated with the upregulation of IgG4 in a patient or subject by administering to the patient or subject a therapeutically effective amount of the agent. The disease may be selected from the Markush group consisting of pemphigus vulgaris, pemphigus foliaceus, anti-LGI1 encephalitis, and MuSK myasthenia gravis. [0031] In another embodiment, the invention provides a composition including the agent as a first compound and a second compound (an at least one additional agent) comprising a moiety capable of conjugating to the IgG4-binding moiety of the first compound to form the first compound. [0032] Several objects, features, aspects, and advantages of the invention will become more apparent from the following detailed description of embodiments of the invention, along with the accompanying drawings.

7 30123-WO-PCT BRIEF DESCRIPTION OF THE DRAWINGS [0033] For illustration, some embodiments of the invention are shown in the drawings described below. Like numerals in the drawings indicate like elements throughout. The invention is not limited to the precise arrangements, dimensions, and instruments shown. [0034] FIG.1 shows the structure of one embodiment of the bifunctional composition of matter (agent). [0035] FIG.2 is a comparison of sequences 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 in IgG2 and IgG4 from IgG1 are noted. The hinge and lower hinge regions, with the N-glycosylation site, N297, are noted. Numbering is according to EU numbering scheme. Sequences important for FcgR-binding, C1q, and FcRn are noted by shading. Some increased ADCC mutants are shown above or below the sequences, as noted: Xencor’s S239D, A330L, I332E; Genentech’s S298A, E333A, K334A; and Eli Lilly/AME’s P247I, A339D/Q. Examples of FcRn-binding mutants for prolongation of half-life are also shown, as noted: MedImmune’s YTE mutant (M252Y, S254T, T256E), PDL’s T250Q, M428L mutant, Sally Ward’s H433K mutant, N434Y mutant, and Genentech’s N434A mutant are shown in boxes. [0036] FIG.3 is a table of R-groups (TABLE 4) for MATE reagents and bifunctional MoDE final compounds. [0037] FIG 4 is a set of schematic drawings for four of the composition of matter embodiments, specifically AGT401, AGT402, AGT403, and AGT404. [0038] FIG.5 is set of chemical structures for some of the composition of matter embodiments, specifically AGT402 in FIG.5A, AGT508 and AGT501 in FIG.5B, AGT502, AGT509, and AGT401 in FIG.5C, and AGT403 and AGT404 in FIG.5D. DETAILED DESCRIPTION OF THE INVENTION [0039] The following detailed description is provided to aid persons having ordinary skill in the biomedical art. Exemplary embodiments are described in detail. However, these embodiments are only exemplary. This disclosure is not limited thereto but rather is defined by the scope of the appended claims. Persons having ordinary skill in the biomedical art may make modifications and variations in the embodiments described in this specification without departing from the spirit or scope of this disclosure.

8 30123-WO-PCT Industrial Applicability [0040] The invention provides a medically useful composition of matter (agent) for treating or slowing down the progression of diseases that are manifestations of IgG4-related diseases (IgG4-RD). IgG4-mediated autoimmune diseases (IgG4-AIDs) establish IgG4 as pharmacological target for degradation their diagnosis, based on clinical symptoms and serum antigen-specific IgG4 autoantibodies. These diseases include pemphigus vulgaris, pemphigus foliaceus, LG1 encephalitis, MuSK (IgG4 myasthenia gravis), type 1 autoimmune pancreatitis, interstitial nephritis, Riedel's thyroiditis, Mikulicz's disease, Küttner's tumor, inflammatory pseudotumors in several 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). Alternative IgG4 Indications include membranous nephropathy, thrombotic thrombocytopenic purpura, chronic inflammatory demyelinating polyneuropathy, and IgG4 related disease. [0041] Whatever area of the body is involved, the hallmark histopathological features of IgG4- related disease are (1) a dense infiltrate of lymphocytes and IgG4-positive plasma cells; (2) fibrosis arranged in a storiform pattern, and phlebitis, where the venous channels are obliterated by the dense infiltrate within both the venous walls and the lumen. See Deshpandeet al., Modern Pathology.25(9), 1181–1192 (May 18, 2012). TABLE 5 IgG4 indication overview. Criteria Pemphigus Pemphigus Anti-LGI1 MuSK myasthenia vulgaris foliaceus encephalitis gravis Patient about 30K 1-9/100,0000. about 2,300 Affecting 5–8% of Population patients. Incidence: 5/1 (USA). Incidence: all MG patients. Incidence: 1-10/ million 0.8/1 million about 4,800 1 million patients in USA

9 30123-WO-PCT TABLE 5 IgG4 indication overview. Criteria Pemphigus Pemphigus Anti-LGI1 MuSK myasthenia vulgaris foliaceus encephalitis gravis Disease Acute and Skin blistering Acute and chronic Acute and chronic; Severity chronic; primarily in more disease: Seizures + usually has an Autoimmune superficial layers of cognitive acute onset blistering epidermis (less severe impairment affecting mainly the disorder than pemphigus facial-bulbar affecting vulgaris) muscles. Rapid mucous progression of membrane and symptoms the skin Unmet Need High. Current Moderate High. Current Moderately high. A standard of care standard of care severe form of the rituximab has a relies on steroid disease has bulbar long onset of use. impairment usually act. Prolonged associated with use of steroids rapid deterioration, has a multitude frequently leading of severe side to respiratory crisis. effects Pathophysio- Antigenic Antigenic Target: Antigenic Target: Antigenic Target: logy Target: Desmoglein 1. LGI1. LGI1-IgG (a MuSK. Desmoglein 3, blockade of component of the Characterized by blockade of desmoglein trans-synaptic antibodies desmoglein resulting in loss of complex) targeting the resulting in loss cell–cell associated with muscle-specific of cell–cell interactions leading limbic encephala- kinase (MusK) interactions to skin blistering. itis, seizures and resulting in leading to skin cognitive various neurologic blistering. impairment. symptoms. Scientific Proven Proven Proven Proven Rationale Pathogenicity of Pathogenicity of Pathogenicity of Pathogenicity of IgG4 IgG4. Passive LGI1 IgG4. Antibodies transfer of IgG4 autoantibodies disrupt normal antibodies could signaling in NMJ in reproduce the murine models disease in animals [0042] Pemphigus vulgaris is an autoimmune blistering disorder affecting mucous membrane and the skin where positive functional outcomes can be followed by a visual observation of the improved

10 30123-WO-PCT skin condition follows the depletion of IgG4 antibodies. There is a relatively large patient population, a 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 a loss of cellular adhesion. Biomedical data show that the decreases in autoantibody titer are associated with clinical response. TABLE 6 Target product profile for pemphigus. Acceptable case Better acceptable case Aspirational case Mechanism of action ASGPR-mediated lysosomal degradation of circulating IgG4 antibodies including those that induce pathogenicity in IgG4-autoimmune diseases (IgG4-AID), Pemphigus vulgaris or foliaceus. Technology Molecular degraders of extracellular proteins: Bispecific molecule composed approach of ASGPR targeting molecule linked to either an anti-IgG4 VHH or IgG4- binding peptide. Indication/patient Indicated for chronic treatment of IgG4 autoimmune diseases. The initial population indication is patients eighteen years and older with confirmed diagnosis of pemphigus vulgaris or foliaceus with anti-Dsg1 or Dsg3 IgG4 antibodies. Broader indications are patients eighteen years and older with IgG4 AID (MuSK-MG, CIDP, anti-LGI1 encephalitis) Clinical efficacy Endpoints: Low number of adverse events and no serious adverse events. profile Reduction in skin lesions. Reduction in pemphigus disease area index (PDAI). Autoimmune blister quality of life (ABQOL score). Time to complete clinical remission (CR). Biomarker Serum levels of total Serum levels of total Serum levels of total measurements IgG4 Abs and anti- IgG4 Abs and anti- IgG4 Abs and anti- Dsg1/3 IgG4 Abs Dsg1/3 IgG4 Abs Dsg1/3 IgG4 Abs reduced by >50%. reduced by >70%. reduced by 85-95%. Serum levels of IgG Serum levels of IgG Serum levels of IgG subtypes 1, 2 and 3 subtypes 1, 2 and 3 subtypes 1, 2 and 3 remain unchanged. remain unchanged. remain unchanged. Safety & tolerability Well tolerated with Well tolerated with Well tolerated with profile favorable safety profile. favorable safety profile. favorable safety No drug hypersensitivity No drug profile. No drug or immunogenicity hypersensitivity or hypersensitivity or Discontinuations due to immunogenicity. immunogenicity. AEs <15%. Discontinuations due to Discontinuations due AEs <10%. to AEs <5%.

11 30123-WO-PCT TABLE 6 Target product profile for pemphigus. Acceptable case Better acceptable case Aspirational case Route of Intravenous injection, Subcutaneous or IV Subcutaneous or IV administration/ biweekly. injection, biweekly. injection, monthly, dosing regimen top-up therapy as indicated. [0043] Muscle-specific kinase (MuSK)-positive myasthenia gravis a distinct and often more severe form of myasthenia gravis often characterized by bulbar features, and which has a neurological indication. There is a correlation between autoantibody titer and clinical outcomes. IgG4 antibodies but not IgG1-3 from inhibit agrin-dependent binding between MuSK and Lrp4. IgG4 antibodies from MuSK MG patients cause disease without requiring complement. IgG4 autoantibodies to MuSK block its interaction with LRP4, thereby obstructing a key trophic signaling cascade at the neuromuscular junction and resulting in inhibited clustering of acetylcholine receptors leading to muscle weakness. [0044] MuSK myasthenia gravis (MuSK MG) is an indication for the therapeutic targeting of IgG4 autoantibodies, with the objective of achieving clinical remission in patients. The pathogenic mechanism in MuSK myasthenia gravis involves IgG4 autoantibodies with differing rates of re-synthesis compared to antigenic IgG4. This slower recovery of the pathogenic process underscores the need for sustained and targeted therapeutic interventions. TABLE 7 Target product profile for MuSK-myasthenia gravis. Acceptable case Better acceptable case Aspirational case Mechanism of ASGPR-mediated lysosomal degradation of circulating IgG4 antibodies, including action those that induce pathogenicity in Muscle-Specific Kinase Myasthenia Gravis (MuSK-MG). Technology Molecular degraders of extracellular proteins: Bispecific molecule composed of approach ASGPR targeting molecule linked to an anti-IgG4 VHH nanobody. Indication/ Indicated for chronic treatment of IgG4 autoimmune diseases. Initial indication is patient with patients eighteen years and older with confirmed diagnosis of refractory population MuSK-MG. Positive IgG4 anti-MuSK antibody test at screening. Broader indications are patients eighteen years and older with IgG4 AID (pemphigus vulgaris, CIDP, anti-LGI1 encephalitis) Clinical efficacy Endpoints. Reduction in clinical symptoms using Qualitative MG (QMG), and MG profile Activities of Daily Living (MG-ADL), MG Composite (MGC) and MG Quality of Life (MG-QoL-15r).

12 30123-WO-PCT TABLE 7 Target product profile for MuSK-myasthenia gravis. Acceptable case Better acceptable case Aspirational case Key biomarker Serum levels of total Serum levels total IgG4 Serum total IgG4 Abs measures IgG4 Abs and anti-MuSK Abs and anti-MuSK IgG4 and anti-MuSK IgG4 Abs IgG4 Abs reduced by Abs reduced by >70% reduced by 85-95% >50%. Serum levels of Serum levels of IgG Serum levels of IgG IgG subtypes 1, 2 and 3 subtypes 1, 2 and 3 subtypes 1, 2 and 3 remain unchanged. remain unchanged. remain unchanged. Safety & Well tolerated with Well tolerated with Well tolerated with tolerability favorable safety profile. favorable safety profile. favorable safety profile. profile No drug No drug hypersensitivity No drug hypersensitivity hypersensitivity or or immunogenicity or immunogenicity immunogenicity Discontinuations due to Discontinuations due to Discontinuations due to adverse events <10%. adverse events <5%. adverse events <15%. Route of Intravenous injection, Subcutaneous or IV Subcutaneous or IV administration/ biweekly. injection, biweekly. injection, monthly – dosing regimen top-up therapy as indicated. [0045] Anti-LGI-1 encephalitis is a rare acute IgG4-related disease with a fast readout, including seizures and cognitive impairment. This disease is treated with rituximab, but this medicine takes a relatively long time to act, so there is a need for an earlier treatment. [0046] Patients with anti-leucine-rich glioma inactivated 1 (LGI1) encephalitis develop memory disturbances, confusion, and faciobrachial dystonic seizures. LGI1 autoantibodies block the binding of LGI1 to ADAM22, reducing AMPA receptor (AMPAR) levels postsynaptically, resulting in neuronal hyperexcitability. Ariño et al., Neurology, 87(8), 759-65 (August 23, 2016) showed that of fifty-seven LGI1 patients with sera available for antibody subclass analysis, all showed IgG4 Abs. TABLE 8 Target product profile for diseases. Criteria CIDP IgG4 Related Disease TTP Membranous Nephropathy Patient Prevalence 1 to Prevalence about 60 / 1 Prevalence: 10/1 about 23,000 Cases population 9/100,000, about million; about 19,000 million; about 3,000 USA. Incidence: about 16,000 (USA). (USA). Incidence 0.3-1.1 (USA). Incidence: 1 10/1 million Globally Incidence: per 100,000 case/million 0.33/100,000

13 30123-WO-PCT TABLE 8 Target product profile for diseases. Criteria CIDP IgG4 Related Disease TTP Membranous Nephropathy Disease Mild to severe; Chronic, relapsing Often considered Chronic disease: Can severity characterized by remitting disease often acute; some cause significant muscle weakness, with systemic multiorgan patients require proteinuria and fatigue, motor involvement; life chronic treatment nephrotic syndrome. function loss, expectancy after diagnosis Microangiopathic Can progress to sensory changes is about 20 years hemolytic anemia; kidney failure in risk of severe cases. neurocognitive deficits and cardiovascular events Unmet Moderate High Mild-moderate High need Patho- Antigenic Target: Systemic fibrosing Antigenic Target: Antigenic Target: physiology CNTN1, NF155. characterized by dense ADAMTS13. PLA2R, THSD7A. Blockade of NF155– infiltration of IgG4-positive Blockade of binding Antibodies against the CNTN1 interaction, plasma cells in tissue with and cleavage of phospholipase A2 resulting in loss of or without elevation in vWF, resulting in receptor (PLA2R) on connectivity plasma multimers that podocytes. between cells and cause impaired microthrombi. neuroconduction. Scientific Proven IgG4 autoantibodies may Proven Likely Pathogenicity of rationale Pathogenicity of not be directly pathogenic Pathogenicity of IgG4. Challenging IgG4 in passive IgG4 animal models transfer models. The because rodent mechanism still not podocytes do not fully understood. express PLA2R IgG3 also likely contributory

14 30123-WO-PCT TABLE 8 Target product profile for diseases. Criteria CIDP IgG4 Related Disease TTP Membranous Nephropathy Clinical Risk of relapse. Primary – Time to flare Platelet count Proteinuria reduction, endpoints INCAT, I-RODS and through week 52. recovery. renal function grip strength. Annualized flare rate. Resolution of Proportion with flare-free hemolysis and remission over time relapse rates. Previously recommended treatments for IgG4-related diseases. [0047] In untreated patients with active disease, the previously recommended first-line agent for induction of remission is glucocorticoids, e.g., prednisolone 30–40 mg per day for two–four weeks, then gradually tapered over three to six months, unless contraindications exist. Glucocorticoids characteristically result in a rapid improvement in clinical features and often a resolution of radiographic features. When advanced fibrotic lesions have resulted in irreversible damage, the response to glucocorticoids and other current treatment options may be poor or even absent. Definitions [0048] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are listed below. As used in this application, except as otherwise provided in this specification, each of these terms shall have the meaning set forth below. Additional definitions are set forth throughout the application. Where a term is not specifically defined in this specification, that term is given a biomedical art-recognized meaning applying that term in context to its use in describing the invention. These definitions aid in describing embodiments but are not intended to limit the claimed invention. [0049] The articles "a" and "an" have the plain meaning of one or to more than one, i.e., at least one, of the grammatical object of the article unless the context indicates otherwise. For example, "an element" means one element or more than one element. [0050] The term “ABT” has the biomedical art-recognized meaning of an antibody-binding moiety. In some embodiments of this specification, the ABT binds to an IgG4 antibody. [0051] The term “active Ingredient” has the United States Food & Drug Administration-provided meaning of any component that provides pharmacological activity or other direct effect in the diagnosis,

15 30123-WO-PCT cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of a human body or an animal body. [0052] The term “ADCC” has the biomedical art-recognized meaning of antibody-dependent cell- mediated cytotoxicity. ADCC is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system kills a target cell, whose membrane-surface antigens have been bound by specific antibodies. [0053] The term “ADCP” has the biomedical art-recognized meaning of antibody-dependent cell- mediated phagocytosis, an immunological mechanism of elimination whereby tumor cells are targeted with antibodies to promote their clearance from the body by phagocytic immune cells. [0054] The term “AF488” has the biomedical art-recognized meaning of Alexa Fluor 488, a bright, green-fluorescent dye with excitation suited for the 488 nm laser line and is used for imaging and flow cytometry. [0055] The term “AF647” has the biomedical art-recognized meaning of the far-red dye Alexa Fluor 647, which has an excitation peak at 650 nm and an emission peak at 665 nm. Alexa 647 is useful for flow cytometry, microscopy, and super-resolution microscopy. [0056] The term “agent” has the biomedical art-recognized meaning of a composition of matter useful for performing a function. Several useful biomedically functions are described in this specification. [0057] The term “alleviate” has the biomedical art-recognized meaning of a process by which the severity of a sign or symptom of a disorder is reduced. A sign or symptom can be alleviated without being eliminated. The administration of compositions or pharmaceutical compositions of the invention may or can lead to the elimination of a sign or symptom, however, elimination is not required. Effective dosages should be expected to decrease the severity of a sign or symptom. [0058] The term “an effective amount” and the term “a therapeutically effective amount” has the biomedical art-recognized meaning of an amount effective to achieve its intended purpose. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend on the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a situation can be determined by routine experimentation that is within the skill and judgment of the clinician. In embodiments, the disease or condition to be treated is tendinopathy. [0059] The term “anti-IgG4 antibody” has the biomedical art-recognized meaning of an antibody that selectively binds an IgG4 antibody.

16 30123-WO-PCT [0060] The term “antigen-binding fragment thereof” has the biomedical art-recognized meaning of (1) a fragment of an intact antibody that binds to the same antigen recognized by the full-length antibody, such as F(abʹ)2, F(ab)2, Fabʹ, Fab, Fv, sFv, or other fragments consisting of the variable regions, or (2) any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. The term antigen-binding portion of an antibody encompasses single chain antibodies. [0061] The term “asialoglycoprotein receptor (ASGPR) binding group” has the biomedical art- recognized meaning of a binding group which binds to a hepatocyte asialoglycoprotein receptor. The ASGPR-binding group selectively binds to hepatocyte asialoglycoprotein receptor on the surface of hepatocytes. In several embodiments of this specification, an ASGPR-binding group is a component of a bifunctional agent as a cellular receptor-binding moiety which is covalently bound to the IgG4-binding moiety through a linker group or directly. It is through this ASGPR moiety that bifunctional agents complexed with a circulating protein, e.g., IgG4 antibody, bind to hepatocytes. After the bifunctional agent complexed with a circulating protein is bound to a hepatocyte or other cell, the circulating protein is taken into the hepatocyte or other cell via a phagocytosis mechanism, wherein the circulating protein is degraded through lysosomal degradation. [0062] The term “asialoglycoprotein receptor (ASGPR) has the biomedical art-recognized meaning of lectins which bind asialoglycoprotein and glycoproteins from which a sialic acid has been removed to expose galactose residues. These cellular receptors are located on mammalian hepatocytes and other cells, such as glandular cells of the gallbladder and the stomach. ASGPR remove target glycoproteins from circulation. [0063] The term "at least one of," when preceding a list of elements, modifies the entire list of elements and does not modify the individual elements of the list. [0064] The term “AT” has the biomedical art-recognized meaning of an antibody moiety. In some embodiments of this specification, the AT binds to IgG4. [0065] The term “bafilomycin” (bafilomycin A1) has the biomedical art-recognized meaning of a macrolide antibiotic that inhibits vacuolar H+-ATPase (V-ATPase). It's used in cell biological assays to block autophagy and reduce cell growth in cancer cell lines. [0066] The term “cellular receptor-binding moiety” has the biomedical art-recognized meaning. In several embodiments of this specification, the cellular receptor-binding moiety is an asialoglycoprotein receptor (ASGPR) binding group.

17 30123-WO-PCT [0067] The term “cellular receptor” has the biomedical art-recognized meaning of a protein on the surface of a cell that binds to a compound, e.g., a ligand, e.g., a protein, in solution or on another cell. Generally, ligand-receptor-binding induces one or more biological responses. In this specification, an asialoglycoprotein receptor (ASGPR) is a cellular receptor on the surface of hepatocytes or other cells that binds to an asialoglycoprotein or a derivative thereof. [0068] The term “chemistry, manufacturing, and controls” (pharmaceutical quality, CMC) has the biomedical art-recognized meaning of the several procedures used to assess the physical and chemical characteristics of drug products, to ensure their quality and consistency during manufacturing, and to prepare data for submissions to regulatory authorities. [0069] The term “chimerized” has the biomedical art-recognized meaning. Chimeric antibodies are made by fusing variable domains from one species, such as a mouse, with constant domains from another species, such as a human being. With such biotechnical manipulation, chimeric antibodies retain the foreign antibody’s antigen specificity and affinity. [0070] The term “combination therapy” and the “co-therapy” has the biomedical art-recognized meaning of the administration of a composition described in this specification and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co- action of these therapeutic agents. The beneficial effect of the combination may include, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time, usually minutes, hours, days, or weeks depending on the combination selected. The term combination therapy” includes the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies, e.g., surgery or radiation treatment. Where the combination therapy further comprises a non-drug treatment, the non- drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents a is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks. [0071] The term “complementarity determining region (CDR)” has the biomedical art-recognized meaning of a polypeptide region of an antibody heavy chin or an antibody light chain that is a determinant of the antibody to antigen-binding. Each antibody heavy chain has three CDRs. Each antibody light chain has three CDRs, usually different from the three CDRs on an antibody heavy chain. Persons having ordinary skill in the biomedical art calculate the using a standardized numbering method

18 30123-WO-PCT known as the Kabat numbering scheme. Kabat et al, (1991) Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, MD., USA), although other numbering schemes such as Chothia and IMGT are also used by persons having ordinary skill in the biomedical art. [0072] The term "comprises," the term "comprising," the term "includes," and the term "including" specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof. [0073] The term “desmoglein 3” (Dsg3) has the biomedical art-recognized meaning of a gene that encodes a member of the cadherin family of proteins. Desmoglein 3 multiprotein complexes are involved in cell adhesion, cytoskeletal structural organization, and cell signaling. Mice lacking Dsg3 exhibit loss of keratinocyte cell adhesion, which results in a blistering phenotype observed in pemphigus vulgaris patients. Binding of antibodies against Dsg3 leads to loss of intracellular adhesion and results in intraepithelial blister formation. The overall homology of Dsg3 between mice and humans are 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). Binding of monoclonal antibodies against Dsg3 on epidermal keratinocytes results in a loss of intercellular adhesion and formation of intraepithelial blisters (symptoms of pemphigus vulgaris). [0074] The term “DMPK” has the biomedical art-recognized meaning of drug metabolism and pharmacokinetics, which is a discipline that helps identify drugs that are likely to be suitable for further development. [0075] The term “drug product” has the United States Food & Drug Administration definition described in 21 C.F.R. § 314.3 of a finished dosage form, e.g., tablet, capsule, or solution, that contains a drug substance, generally, but not necessarily, in association with one or more other ingredients. [0076] The term “drug substance” has the United States Food & Drug Administration definition described in 21 C.F.R. § 314.3 of an active ingredient that is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of the human body but does not include intermediates used in the synthesis of such ingredient. [0077] The term “Fc-III-4c” has the biomedical art-recognized meaning of a polypeptide region in the fragment crystallizable region (Fc region), the tail region, of an antibody.

19 30123-WO-PCT [0078] The term “Fc-M” has the biomedical art-recognized meaning of a polypeptide region in the fragment crystallizable region (Fc region), the tail region, of an antibody. [0079] The term “FcB-1” has the biomedical art-recognized meaning of a polypeptide region in the fragment crystallizable region (Fc region), the tail region, of an antibody. [0080] The term “FcB-2” has the biomedical art-recognized meaning of a polypeptide region in the fragment crystallizable region (Fc region), the tail region, of an antibody. [0081] The term “first,” “second,” “third,” etc. have the plain meaning of describing several elements, components, regions, layers, and/or sections, these elements, components, regions, layers, and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. A first element, component, region, layer, or section could be called a second element, component, region, layer, or section without departing from the teachings of the present embodiments. [0082] The term “Good Laboratory Practice” has the United States Food & Drug Administration definition described in 21 C.F.R. Part 58, Good Laboratory Practice for Nonclinical Studies. [0083] The term “hepatocyte” has the biomedical art-recognized meaning of a cell of the main parenchymal tissue of the liver. Hepatocytes make up 55-65% of the liver's mass. [0084] The term “humanized” has the biomedical art-recognized meaning a protein, e.g., an antibody, is genetically engineered so it closely resembles the polypeptide structure of the human homologue. A variable domain of an antibody of rodent origin can be fused to a constant domain of human origin, thus retaining the specificity of the rodent antibody. The human origin domain need not originate directly from a human in that it is first synthesized in a human. Instead, human domains can be generated in rodents whose genome incorporates human immunoglobulin genes. The antibody can be partially or completely humanized. In one approach, there are four general steps used to humanize a monoclonal antibody, These are (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable domains; (2) designing the humanized antibody, i.e., deciding which antibody framework region to use during the humanizing process; (3) the actual humanizing methodologies/techniques; and (4) the transfection and expression of the humanized antibody. [0085] The term “IC₅₀“ has the biomedical art-recognized meaning of an amount, concentration, or dosage of a particular test compound that achieves a 50% inhibition of a maximal response in an assay that measures such response.

20 30123-WO-PCT [0086] The term “IgG” antibody has the biomedical art-recognized meaning. Each IgG molecule consists of the basic four-chain immunoglobulin structure—two γ (gamma) heavy chains and two identical light chains (either kappa or lambda)—and carries two identical antigen-binding sites. There are four subclasses of IgG, each with minor differences in its H chains but with distinct biological properties. [0087] The term “IgG1” antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-1 chain C region is a protein that in humans is encoded by the IGHG1 gene. [0088] The term “IgG2” antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-2 chain C region is a protein that in humans is encoded by the IGHG2 gene. [0089] The term “IgG4-binding moiety” has the biomedical art-recognized meaning a moiety on a binding protein, e.g., and IgG antibody or a fragment thereof, that binds to an IgG4 antibody. [0090] The term “IgG4” antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-4 chain C region is a protein that in humans is encoded by the IGHG4 gene. IgG4 has little effector function. IgG4 cannot fix complement. [0091] The term “IVIG” has the biomedical art-recognized meaning of the administration of intravenous immunoglobulin (IVIG).^{[0092] The term “K}_{D” has the biomedical art-recognized meaning of the measured equilibrium} dissociation constant between a compound or ligand and a protein or binding domain of a protein. [0093] The term “LGI1” has the biomedical art-recognized meaning of a neuronal antigen involved in synaptic function as it is part of the voltage-gated potassium channel complex and is involved in linking transmembrane proteins across the synaptic cleft. [0094] The term “linker moiety” has the biomedical art-recognized meaning of a moiety of a chemical compound that links one moiety of the chemical compound to another moiety of the same compound. In several embodiments of this invention, the linker moiety connects an anti-IgG4 IgG antibody moiety to a cellular receptor-binding moiety, [0095] The term “MoDE” has the proprietary meaning of molecular degraders. See Intl. Pat. Publ. WO 2019/199634 (Yale University) and Intl. Pat. Publ. WO 2019/199621 (Yale University). [0096] The term “moiety” has the biomedical meaning of a defined chemical group or entity with a particular structure or activity. A moiety generally refers to a part of a molecule, e.g., in an ester RCOOR’, the alcohol moiety is RO−. In some embodiments, a moiety of an agent, e.g., a target agent, a peptide agent, an antibody agent, etc. retains one or more or all desirable structural features, properties, functions, and/or activities of a compound. In some embodiments, a cellular receptor-

21 30123-WO-PCT binding moiety can bind to a target, optionally in a comparable fashion, as its corresponding target- binding agent; in some embodiments, a target agent moiety maintains one or more desired structural features, properties, functions, and/or properties comparable to its corresponding target agent; in some embodiments, an antibody agent moiety maintains one or more desired structural features, properties, functions, and/or properties, e.g., 3-dimension structure, antigen specificity, antigen-binding capacity, and/or immunological functions, etc. comparable to its corresponding antibody agent. In some embodiments, a moiety of an agent, e.g., a target agent moiety, a peptide agent moiety, an antibody agent moiety, etc. is a monovalent (for a monovalent moiety), bivalent (for a bivalent moiety), or polyvalent (for a polyvalent moiety) radical of an agent, e.g., a target agent (for a target agent moiety), a peptide agent (for a peptide agent moiety), an antibody agent (for an antibody agent moiety), etc. In some embodiments, a monovalent radical is formed by removing a monovalent part, e.g., hydrogen, halogen, another monovalent group like alkyl, aryl, etc. from a compound/agent. In some embodiments, a bivalent or polyvalent radical is formed by removing one or more monovalent, e.g., hydrogen, halogen, monovalent groups like alkyl, aryl, etc., bivalent and/or polyvalent parts from a compound/agent. In some embodiments, radicals are formed by removing hydrogen atoms. In some embodiments, a moiety is monovalent. In some embodiments, a moiety is bivalent. In some embodiments, a moiety is polyvalent. [0097] The term “monotherapy” has the biomedical art-recognized meaning of the administration of a single active or therapeutic compound to a subject. Monotherapy usually is the administration of a therapeutically effective amount of an active composition. [0098] The term “Multimodal Antibody Therapy Enhancers” (MATE or MATES) has the proprietary meaning. See Intl. Pat. Publ. WO 2021/102052 (Kleo Pharmaceuticals). [0099] The term “nanobody” has the biomedical art-recognized meaning. [00100] The term “antibody” refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds an antigen. The term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms including full length antibodies and antigen- binding portions thereof; including, e.g., an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fabʹ, a F(abʹ)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a nanobody, a multi-specific (e.g., trispecific) antibody, a dual specific antibody, a bispecific antibody, an anti-idiotypic antibody, a functionally active epitope-binding portion thereof, and/or bifunctional hybrid antibodies.

22 30123-WO-PCT [00101] The term "on" has the plain meaning. When an element is referred to as being on another element, it can be directly in contact with the other element or intervening elements may be present therebetween. When an element is referred to as being "directly on" another element, there are no intervening elements present. [00102] The term "or" as used in this specification means "and/or." The term "and/or" as used in this specification includes any and all combinations of one or more of the associated listed items. [00103] The term “other degrading cells” has the biomedical art-recognized meaning. Asialoglycoprotein receptors (ASGPRs) are on the glandular cells of the gallbladder and the stomach. [00104] The term “partially humanized” has the biomedical art-recognized meaning a protein, e.g., an antibody, is genetically engineered so it more closely resembles the polypeptide structure of the human homologue. A variable domain of an antibody of rodent origin can be fused to a constant domain of human origin, thus retaining the specificity of the rodent antibody. The domain of human origin need not originate directly from a human in that it is first synthesized in a human. Instead, human domains can be generated in rodents whose genome incorporates human immunoglobulin genes. The antibody can be partially or completely humanized. [00105] The term “pharmaceutically acceptable excipient” has the biomedical art-recognized meaning of an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use or human pharmaceutical use. A “pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient. A thorough discussion of pharmaceutically acceptable excipients is available in Remington’s, Pharmaceutical Sciences 23rd edition (Elsevier, 2020). [00106] The term “pharmaceutically acceptable” has the biomedical art-recognized meaning of those compounds, anions, cations, materials, compositions, carriers, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. [00107] The term “protein-binding moiety” has the biomedical art-recognized meaning of a region of a chemical composition, e.g., a polypeptide region of a chemical composition, that specifically binds to a protein, e.g., a specific protein. [00108] The term “rIgG” has the biomedical art-recognized meaning of recombinant human IgG.

23 30123-WO-PCT [00109] The term “ROC” has the biomedical art-recognized meaning of receiver operating characteristic curve. [00110] The term “subject” and the term “patient” have the biomedical art-recognized meanings. The term patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. [00111] The term “TBT” in this specification has the meaning of a target-binding moiety, a cellular receptor-binding moiety. In some embodiments of this specification, the TBT binds to ASGPR. [00112] The term “treating” and the “treat” has the biomedical art-recognized meaning of the management and care of a patient for combating a disease, condition, or disorder, and includes the administration of a composition described in this specification to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder. [00113] The term “universal antibody-binding moiety” has the biomedical art-recognized meaning of a polypeptide region of an antibody-binding protein that binds a class of antibodies, rather than a specific set of antibodies. [00114] The term “VHH” has the biomedical art-recognized meaning of Variable domain of Heavy chain of Heavy-chain antibody derived from camelid antibodies. VHH has nine beta-sheets forming a cylindric structure. Several regions may be involved in binding. Human IgG is about 150 kilodaltons. Camelid IgG is about 90 kilodaltons. VHH single chain domain is about 15 kilodaltons. The most important regions 4>1, 2 (greatest difference in uptake). The least important: regions 3, 5 (very minor difference in uptake). [00115] Some embodiments are described below by referring to structures and schemes, to explain aspects of the description. [00116] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by persons having ordinary skill in the biomedical art. [00117] This specification does not concern a process for cloning humans, methods for modifying the germ line genetic identity of humans, uses of human embryos for industrial or commercial purposes, or procedures for modifying the genetic identity of animals likely to cause them suffering with no substantial medical benefit to humans or animals resulting from such processes. Methods of administering the bifunctional degrader to subjects and measuring the outcomes.

24 30123-WO-PCT [00118] How to identify subjects to whom the bifunctional degrader will be administered. Serum immunoglobulin G4 (IgG4) is often elevated, but this is not always the case. However, IgG4 blood lab levels greater than 135 are considered an evolving diagnostic criterion for disease suspicion. [00119] IgG4 levels vary among healthy individuals and even within an individual patient. In one retrospective cohort study, baseline concentrations of serum IgG4, IgE, and blood eosinophils are independently predictive of relapse risk following treatment. The higher the baseline values, the greater the relapse risk and the shorter the time to relapse. Wallace et al., Rheumatology, 55(6), 1000–1008 (June 6, 2016). [00120] Definite diagnosis is important because treatments for IgG4 disease and other diseases that mimic it differ. Satou et al., Pathol. Int., 70(7), 391–402 (July 2020). While serum IgG4 levels may be suggestive, a definitive histological diagnosis is necessary to confirm IgG4 disease and rule out similar conditions. Chen et al., Haematologica, 104(3), 444 (March 2019). The sensitivity of IgG4 levels in diagnosing IgG4-related disease varies depending on the assay used, the number of affected organs, and possibly the patient’s geographic origin. See Acharya et al.,. J. Community Hosp. Intern. Med. Perspect., 14(3), 113-118 (May 7, 2024). [00121] How to administer. The best mode of administration depends on where treatment is taking place, whether a hospital or outpatient. [00122] The delivery of therapeutic agents can be via subcutaneous injection, e.g., by administering a 6 mL subcutaneous infusion or by further optimizing dose concentration and volume for subcutaneous administration. Alternative formulations, such as a VHH-Fc fusion protein, can reduce the amount needed to be administered. In one embodiment, the Non-Fc fusion VHH molecules present lower- molecular, which can be advantageous for subcutaneous formulation, with an estimated target of about 125 mg dose. [00123] Dividing doses, e.g., administering 125 mg across four doses, can be a method for administering a full 500 mg dose. Alternative formulations could necessitate a loading dose to achieve sufficient IgG4 depletion. [00124] The inventors performed a set of assays in mouse and rat to provide insights into subcutaneous and intravenous administration routes with repeat dose regimens to show the bioavailability to a subject of the agents administered. [00125] Methods of measuring the removal of IgG4 antibody from a patient or subject. Depletion of antibodies leads to functional outcomes. The inventors have performed In vivo depletion assays to measure IgG4 depletion. See the EXAMPLES provided in this specification. Other assays to measure IgG4

25 30123-WO-PCT in the blood circulation and functional outcomes of IgG4 related disease are known to persons having ordinary skill in the biomedical art. The chemical structure of the composition of matter (agent). [00126] In one embodiment, the invention provides a composition of matter (agent) comprising: an IgG4-binding moiety, a cellular receptor-binding moiety which binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a patient or subject, and a linker moiety linking the antibody moiety and the cellular receptor-binding moiety. [00127] In some embodiments, the invention provides a composition of matter (an agent), where the composition of matter has the structure: [CRBM] is a cellular receptor-binding moiety; each [CON] is an optional connector chemical moiety which, when present, connects directly to [CPBM] or to [CRBM] or connects the [LINKER] to [CPBM] or to [CRBM], and [LINKER] is a chemical moiety that covalently attaches to one or more [CRBM] or [CPBM] groups, or a pharmaceutically acceptable salt, stereoisomer, solvate, or polymorph thereof. [00128] In some embodiments, the invention provides a composition of matter (an agent) having a structure selected from the Markush group of structures consisting of: R^CN−(Xaa)y−R^CC, 30123- of. In these structures, a, b, and c may independently be an integer of 1 or greater. In some embodiments, each cellular receptor-binding moiety independently has the structure of −(R^CN−(Xaa)y−R^CC) or salt form thereof. [00129] In some embodiments, the agent has the structure of formula AGN101: wherein: each of a, b and c is independently 1-200; each AT is independently a IgG4-binding moiety; L is a linker moiety; and each TBT is independently a cellular receptor-binding moiety, wherein the IgG4-binding moiety is (1) an anti-IgG4 antibody or an antigen-binding fragment thereof, or (2) anti-IgG4 VHH affinity ligand variant or an antigen-binding fragment thereof. [00130] In some embodiments, the agent has the structure of formula AGN102: wherein: each of a and b is independently 1-200; each AT is independently a IgG4-binding moiety; L is a linker moiety; and each TBT is independently a cellular receptor-binding moiety, wherein the IgG4-binding moiety is (1) an anti-IgG4 antibody or an antigen-binding fragment thereof, or (2) anti-IgG4 VHH affinity ligand variant or an antigen-binding fragment thereof.

27 30123-WO-PCT [00131] In some embodiments, an agent comprises one and no more than one IgG4-binding moiety. In some embodiments, one or no more than one IgG4-binding moiety is bound to a linker moiety. In some embodiments, a is 1. In some embodiments, a is 2 or more. In some embodiments, one and no more than one cellular receptor-binding moiety is bonded to a linker moiety. In some embodiments, b is 1. In some embodiments, two or more cellular receptor-binding moiety is bonded to a single linker moiety. In some embodiments, b is 2 or more. In some embodiments, an agent comprises one and no more than one cellular 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, an agent comprises two or more IgG4-binding moieties. In some embodiments, b is 2 or more and c is 1. In some embodiments, b is 2 or more and c is 2 or more. In some embodiments, b is 1 and c is 2 or more. [00132] 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. Persons having ordinary skill in the biomedical art know that several technologies can conjugate antibody moieties with target-binding moieties, e.g., certain technologies used for preparing antibody-drug conjugates in accordance with this specification. In some embodiments, target-binding moieties are connected to antibody moieties through certain types of groups and/or amino acid residues. In some embodiments, target-binding moieties are connected to lysine residues optionally through linker moieties. In some embodiments, target-binding moieties are connected to cysteine residues optionally through linker moieties. In some embodiments, target-binding moieties are connected to unnatural amino acid residues optionally through linker moieties. In some embodiments, the invention provides technologies for selectively linking target-binding moieties to certain particular amino acid residues optionally through linker moieties. In some embodiments, provided technologies selectively connect target-binding moieties to certain types of amino acid residues, e.g., lysine residues, optionally through linker moieties. In some embodiments, provided technologies selectively connect target-binding moieties to particular sites of antibody moieties optionally through linker moieties. In some embodiments, provided technologies selectively connect target-binding moieties to certain types of amino acid residues at sites optionally through linker moieties. In some embodiments, target-binding moieties are connected to K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, target-binding moieties are connected to K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, target-binding moieties are connected to K239 and K241 of an IgG4 heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, a cellular receptor-

28 30123-WO-PCT binding moiety is connected to a particular amino acid residue or site optionally through a linker. In some embodiments, each cellular receptor-binding moiety is independently connected to a particular amino acid residue or site optionally through a linker. Persons having ordinary skill in the biomedical art know that an antibody agent may comprise more than one sites, e.g., one on each of the more than one chain, e.g., one or each heavy chain. In some embodiments, an antibody moiety comprises two heavy chains and one or both amino acid residues or amino acid residues corresponding thereto are each independently connected to a cellular receptor-binding moiety, optionally through a linker. In some embodiments, one and no more than one is connected. In some embodiments, c is 1. In some embodiments, both are connected. In some embodiments, c is 2. In some embodiments, both target- binding moieties or both linker moieties are the same. [00133] In some embodiments, each cellular receptor-binding moiety in an agent is the same. In some embodiments, each linker moiety connecting a cellular receptor-binding moiety to an antibody moiety is the same. [00134] In some embodiments, the compositions of matter can be selected from among the following compounds: TABLE 9 ID NO. IgG4-binding moiety Cellular receptor binding Linkage moiety AGN401 VHH w/o hinge region; ABT306 βGN3, TBT103 thiosuccinimide AGN402 VHH-Fc fusion, ABT310 βGN3 via MATE, TBT105 MATE conjugation AGN403 VHH with hinge region βGN3, TBT106 sortase AGN404 VHH w/o hinge region βGN3, TBT106 sortase AGN405 F_c-BP2 (pan-IgG TRAPs) βGN3, TBT103 thiosuccinimide AGN406 F_c-BP2 (pan-IgG TRAPs) PR3 AGN501 VHH-G₄S-Cys, ABT502 βGN3, TBT103 thiosuccinimide AGN502 VHH-G₃ECS-Cys, ABT508 βGN3, TBT103 thiosuccinimide AGN505 F_cIII (pan-IgG TRAPs) αGN3, TBT104 AGN506 F_cIII (pan-IgG TRAPs) βGN3, βGN3, TBT103 thiosuccinimide AGN507 F_c-BP2 αGN3, TBT104 AGN508 VHH-G4S-his6-G4S-Cys ABT501 βGN3, TBT103 thiosuccinimide AGN509 ABT504 βGN3, TBT103 thiosuccinimide The IgG4-binding moiety [00135] In some embodiments, IgG4-binding moieties are connected to antibody moieties through certain types of groups or amino acid residues. In some embodiments, IgG4-binding moieties are connected to lysine residues optionally through linker moieties. In some embodiments, IgG4-binding moieties are connected to cysteine residues optionally through linker moieties. In some embodiments,

29 30123-WO-PCT IgG4-binding moieties are connected to unnatural amino acid residues optionally through linker moieties. In some embodiments, the invention provides technologies for selectively linking IgG4-binding moieties to certain amino acid residues optionally through linker moieties. In some embodiments, provided technologies selectively connect IgG4-binding moieties to certain types of amino acid residues, e.g., lysine residues, optionally through linker moieties. In some embodiments, provided technologies selectively connect IgG4-binding moieties to sites of antibody moieties optionally through linker moieties. In some embodiments, provided technologies selectively connect IgG4-binding moieties to certain types of amino acid residues at sites optionally through linker moieties. In some embodiments, IgG4-binding moieties are connected to K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, IgG4-binding moieties are connected to K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, IgG4-binding moieties are connected to K239 and K241 of a heavy chain and amino acid residues corresponding thereto optionally through linker moieties. In some embodiments, a cellular receptor-binding moiety is connected to a particular amino acid residue or site optionally through a linker. In some embodiments, each cellular receptor-binding moiety is independently connected to a particular amino acid residue or site optionally through a linker. [00136] Persons having ordinary skill in the biomedical art know that an antibody agent may comprise more than one site, e.g., one on each of the more than one chain, e.g., one or each heavy chain. In some embodiments, an antibody moiety comprises two heavy chains, and one or both amino acid residues or amino acid residues corresponding thereto are each independently connected to a cellular receptor-binding moiety optionally through a linker. In some embodiments, one and no more than one is connected. In some embodiments, c is 1. In some embodiments, both are connected. In some embodiments, c is 2. In some embodiments, both IgG4-binding moieties or both linker moieties are the same. [00137] In another embodiment, the IgG4-binding moiety is an IgG4-binding antibody or an antigen- binding fragment thereof. [00138] In another embodiment, the IgG4-binding moiety is a variant of ABT301 (SEQ ID NO: 1). [00139] In another embodiment, the IgG4-binding is a full IgG antibody wherein the heavy chain comprises the three CDR regions of the VHH affinity ligand. A full-length human sourced IgG can be native and have variable glycosylation. [00140] In some embodiments, the IgG4-binding moiety comprises a moiety selected from the Markush group consisting of one or more amino acid residues, a peptide moiety, a cyclic peptide moiety,

30 30123-WO-PCT a peptide comprising one or more natural amino acid residues, and a peptide comprising one or more unnatural natural amino acid residues. [00141] The IgG4-binding moiety may be or may comprise ABT101 or a salt form thereof. In some embodiments, ABT101 is an IgG4-binding moiety. Each IgG4-binding moiety in an agent may be of the same IgG4-binding moiety or a salt thereof. The IgG4-binding moiety can be the anti-IgG4 VHH nanobody or an antigen-binding variant thereof. [00142] In some embodiments, ABT101 is a universal antibody-binding moiety. In some embodiments, ABT101 is a universal antibody-binding moiety which can bind to IgG4 antibodies having different Fab regions. In some embodiments, ABT101 is a universal antibody-binding moiety that binds to a Fc region, e.g., the Fc region that binds to an Fc receptor. The VHH nanobody is a universal binder because the IgG4-binding of the VHH nanobody is through the IgG4 Fc. [00143] In one embodiment, the IgG4-binding moiety is human IgG4 Fc Protein, Tag Free, ACRO Biosystems, Catalog # IG4-H5205, which can be produced by HEK293 expression, with amino acids Glu 99-Lys 327. [00144] In some embodiments, the IgG4-binding moiety comprises a moiety selected from the Markush group consisting of one or more amino acid residues, a peptide moiety, a cyclic peptide moiety, a peptide comprising one or more natural amino acid residues, and a peptide comprising one or more unnatural natural amino acid residues. [00145] The IgG4-binding moiety may be or may comprise: or a salt form thereof. [00146] In some embodiments, ABT101 is a IgG4-binding moiety. Each IgG4-binding moiety in an agent may be of the same IgG4-binding moiety or a salt thereof. [00147] Several IgG4-binding moieties can be used in accordance with the teachings of this specification. Some IgG4-binding moieties and technologies for identifying or assessing IgG4-binding moieties are described in Intl. Pat. Publ. WO 2019/023501 and Intl. Pat. Publ. WO 2019/136442, each of which is incorporated in this specification in its entirety by reference. Persons having ordinary skill in the biomedical art know that additional technologies in the biomedical art may be suitable for identifying or assessing IgG4-binding moieties in accordance with this specification. In some embodiments, an IgG4- binding moiety comprises one or more amino acid residues, each independently natural or unnatural.

31 30123-WO-PCT [00148] In some embodiments, a IgG4-binding moiety is or comprises R^c−(Xaa)z− or a salt form thereof, wherein each variable is as described in this specification. In some embodiments, a IgG4- binding moiety is or comprises ABT101 or a salt form thereof, wherein each variable is as described in this specification. [00149] In some embodiments, a protein-binding moiety is or comprises R^c−(Xaa)z− or a salt form thereof, wherein each variable is as described in this specification. In some embodiments, a protein- binding moiety is or comprises ABT101 or a salt form thereof, wherein each variable is as described in this specification. [00150] In some embodiments, a IgG4-binding moiety is or comprises R^c−(Xaa)z− or a salt form thereof, wherein each variable is as described in this specification. In some embodiments, a IgG4- binding moiety is or comprises ABT101 or a salt form thereof, wherein each variable is as described in this specification. In some embodiments, a IgG4-binding moiety is R^c−(Xaa)z− or ABT101 , or a salt form thereof, and is or comprises a peptide unit. [00151] In some embodiments, amino acid residues may form bridges, e.g., connections formed by side chains optionally through linker moieties, e.g., L); for example, as in many polypeptides, cysteine residues may form disulfide bridges. [00152] In some embodiments, a peptide unit comprises an amino acid residue, e.g., at physiological pH about 7.4, positively charged amino acid residue, Xaa^P). In some embodiments, a peptide unit comprises R. In some embodiments, at least one Xaa is R. [00153] Persons having ordinary skill in the biomedical art know that an amino acid residue may be replaced by another amino acid residue having similar properties, e.g., one Xaa^H, e.g., Val, Leu, etc. may be replaced with another Xaa^H, e.g., Leu, Ile, Ala, etc. , one Xaa^A may be replaced with another Xaa^A, one Xaa^P may be replaced with another Xaa^P, one Xaa^N may be replaced with another Xaa^N, one Xaa^L may be replaced with another Xaa^L, etc. [00154] In some embodiments, an IgG4-binding moiety is or comprises an optionally substituted moiety as shown in the table provided in Intl. Pat. Publ. WO 2019/023501, pages 48-60, which table and patent publication are incorporated in this specification by reference. [00155] In some embodiments, useful technologies for developing or assessing antibody-binding moieties are described in, e.g., 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., ACS Omega, 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), e.g., Fabsorbent, triazines, etc.; Kruljec et

32 30123-WO-PCT al., Bioconjugate Chem., 29(8), 2763-2775 (2018), Intl. Pat. Publ. WO 2012/017021, etc., each of which is incorporated in this specification in its entirety by reference. [00156] In some embodiments, an IgG4-binding moiety is an affinity substance described in Intl. Pat. Publ. WO 2018/199337, the affinity substance of each of which is incorporated in this specification by reference. [00157] In some embodiments, an IgG4-binding moiety is or comprises an adapter protein agent, e.g., as described in Hui et al., Bioconjugate Chem., 26, 1456−1460 (2015). In some embodiments, when used in accordance with this specification, adapter proteins do not require reactive residues, e.g., BPA, to achieve one or more or all advantages. [00158] In some embodiments, an antibody-binding moiety can be selected from the following compounds: TABLE 10 ID NO. SEQ ID NO. Protein structure ABT301 SEQ ID NO: 1 VHH nanobody (122 AAs) ABT304 SEQ ID NO: 9 VHH nanobody (122 AAs), G4S linker, C-term Sortase site, His6 tag ABT305 SEQ ID NO: 10 VHH nanobody with truncation of hinge, G4S linker, C-term Sortase site, His6 tag ABT306 SEQ ID NO: 11 VHH nanobody with truncation of hinge, G4S linker, His6 tag, rigid linker-Cys ABT310 SEQ ID NO: 12 VHH nanobody with truncation of hinge, G4S linker, hIgG1 hinge (C220A)-human IgG1 Fc-L253A/L254A/P329A ABT311 SEQ ID NO: 32 VHH nanobody (122 AAs), G4S linker, His6 tag ABT312 SEQ ID NO: 33 VHH nanobody with truncation of hinge, G4S linker, His6 tag ABT313 SEQ ID NO: 34 VHH nanobody with truncation of hinge, G4S linker, His6 tag, G2S linker, AviTag ABT501 SEQ ID NO: 59 VHH nanobody-G4S-his6-G4S-Cys ABT502 SEQ ID NO: 64 VHH nanobody-G4S-Cys ABT503 SEQ ID NO: 63 VHH nanobody-G3ECS-Cys ABT504 SEQ ID NO: 65 VHH nanobody-G4S-his6-Cys Characterizing antibody-binding moieties. [00159] Many technologies are available for identifying, assessing, or characterizing antibody- binding moieties, e.g., those described in Intl. Pat. Publ. WO 2019/023501, the technologies of which are incorporated in this specification by reference. In some embodiments, an antibody-binding moiety is a moiety, e.g., small molecule moiety, peptide moiety, nucleic acid moiety, etc., that can selectively bind to IgG, and when used in provided technologies can provide and/or stimulate ADCC and/or ADCP. In some embodiments, peptide display technologies, e.g., phase display, non-cellular display, etc., can

33 30123-WO-PCT identify antibody-binding moieties. In some embodiments, an antibody-binding moiety is a moiety, e.g., small molecule moiety, peptide moiety, nucleic acid moiety, etc., that can bind to IgG and optionally can compete with known antibody binders, e.g., protein A, protein G, protein L, etc. [00160] Persons having ordinary skill in the biomedical art know that antibodies of several properties and activities, e.g., antibodies recognizing different antigens, having optional modifications, etc., may be targeted by antibody-binding moieties described in this specification. In some embodiments, such antibodies include antibodies administered to a subject, e.g., for therapeutic purposes. In some embodiments, antibody-binding moieties described in this specification may bind antibodies toward different antigens and are useful for conjugating moieties of interest with several antibodies. [00161] In some embodiments, an IgG4-binding moiety is or comprises a meditope agent moiety. In some embodiments, a meditope agent is described in, e.g., U.S. Pat. Publ.2019/0111149. [00162] In some embodiments, an IgG4-binding moiety can bind to human IgG. In some embodiments, an IgG4-binding moiety can bind to an antibody selected from the Markush group of antibodies consisting of rabbit IgG, IgG1, IgG2, IgG3, and IgG4. In some embodiments, an IgG4-binding moiety binds to IgG1, IgG2, and IgG4. [00163] In some is used in a reference technology as a non- antibody-binding moiety. In some group used in a reference technology as a non-antibody-binding moiety is selected from the Markush group of chemical groups consisting of CH₃−, CH₃C(O)−, CH₃C(O)NH−, CH₃C(O)NHCH₂−, CH₃CH₂−, CH₃CH₂NH−, and CH₃CH₂NHC(O)−. [00164] In some embodiments, antibody-binding moieties, e.g., antibody-binding moieties) bind to targets, e.g., antibody agents for antibody-binding moieties) with a K_D that is about 1 mM^-1 pM or less. In some embodiments, a K_D is about 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 less. In some embodiments, K_D is an affinity selected from the Markush group of affinities consisting of 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

34 30123-WO-PCT less, about 5 nM or less, about 2 nM or less, and about 1 nM or less. In some embodiments, antibody- binding moieties bind to IgG antibody agents with K_D described in this specification. [00165] Persons having ordinary skill in the biomedical art know that antibodies of several properties and activities, e.g., antibodies recognizing different antigens, having optional modifications, etc., may be recruited by antibody-binding moieties described in this specification. In some embodiments, such antibodies include antibodies administered to a subject, e.g., for therapeutic purposes. In some embodiments, antibodies recruited by antibody-binding moieties comprise antibodies toward different antigens. In some embodiments, antibodies recruited by antibody-binding moieties comprise antibodies whose antigens are not present on the surface or cell membrane of target cells. In some embodiments, antibodies recruited by antibody-binding moieties comprise antibodies not targeting antigens present on surface or cell membrane of targets. In some embodiments, antigens on surface of target cells may interfere with the structure, conformation, and/or one or more properties and/or activities of recruited antibodies which bind such antigens. In some embodiments, recruited antibodies are those in IVIG. In some embodiments, IVIG may be administered before, concurrently with or subsequently to an agent or composition. Antibodies of several types of immunoglobulin structures may be recruited. In some embodiments, one or more subclasses of IgG are recruited. In some embodiments, recruited antibodies are selected from the Markush group of antibody classes consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, recruited antibodies are or comprise IgG1 and IgG2. In some embodiments, recruited antibodies are or comprise IgG1, IgG2 and IgG4. In some embodiments, recruited antibodies are or comprise IgG1, IgG2, IgG3 and IgG4. Recruited antibodies may interact several types of receptors, e.g., those expressed by several types of immune cells. In some embodiments, recruited antibodies can effectively interact several types of Fc receptors and provide desired immune activities. In some embodiments, recruited antibodies can recruit immune cells. In some embodiments, recruited antibodies can effectively interact with hFcγRIIIA. In some embodiments, recruited antibodies can effectively interact with hFcγRIIIA on macrophages. In some embodiments, macrophages are recruited to provide ADCC and/or ADCP activities toward a virus, e.g., a SARS-CoV-2 virus, and/or cells infected thereby. In some embodiments, NK cells are recruited to provide immune activities. In some embodiments, recruited antibodies can effectively interact with hFcγRIIA. In some embodiments, recruited antibodies can effectively interact with hFcγRIIA on dendritic cells. In some embodiments, antibody moieties in agents of this specification comprise one or more properties, structures and/or activities of recruited antibodies described in this specification.

35 30123-WO-PCT Fc mutation strategies to enhanced FcRn-binding. [00166] There are many publicly available approaches and mutation sets to increase binding of an Fc to FcRn. See TABLE 11. An older summary was provided by Strohl, Optimization of Fc-mediated effector functions of monoclonal antibodies. Current Opinion in Biotechnology, 20(6), 685-691 (2009). TABLE 11 Examples of Fc sequence engineering for modification of half-life Function Company or source Mutations or changes Increased half-life Medlmmune lgG1·M252Y. S254T, T256E Increased half-life Protein Design Labs lgG1- T250Q, M428L Increased half-life Genentech lgG1-N434A Increased half-life Sally Ward lgG1·H4J3K, N434Y Increased half-life Deny Roopenian lgG1-T307A.E380A,N434A Increased half-life Xeocor Xtend™ technology Lowered endogenous IgG Sally Ward lgG1-M252 Y, S254T,T256E, H433K, N434F, 436H Decreased half-life Deny Roopenian lgG1·1253A Decreased half-life Ell Lilly lgG1-P2571, N434H or Q376V, N434H [00167] For an example of introducing a YTE region in a 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 combining 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). Additional references with details of engineering also available. [00168] For an example of introducing a LS region in a protein (M428L/N434S). See Zalevsky et al. Nature Biotechnology, 28(2), 157-159 (2010).

36 30123-WO-PCT Linker moieties and amino acids thereof [00169] In some embodiments, the linker moiety has the structure of formula LNK101: NH(R^a1)−L^a1−C(R^a2)(R^a3)−L^a2−COOH [LNK101] or a salt thereof, wherein: each of R^a1, R^a2, R^a3 is independently −L^a−R’; each of L^a1 and L^a2 is independently L^a; each L^a is independently a covalent bond, or an optionally substituted bivalent group selected from C₁-C₂₀ aliphatic or C₁-C₂₀ heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced 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−; each −Cy− is independently an optionally substituted bivalent monocyclic, bicyclic, or polycyclic group wherein each monocyclic ring is independently selected from a C_3-20 cycloaliphatic ring, a C_6-20 aryl ring, a 5-20-membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20-membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; each R’ is independently −R, −C(O)R, −CO2R, or −SO2R; each R is independently −H, or an optionally substituted group selected from C_1-30 aliphatic, C_1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, and silicon, C_6-30 aryl, C_6-30 arylaliphatic, C_6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30-membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30-membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or: two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30-membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30-membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently

37 30123-WO-PCT selected from oxygen, nitrogen, sulfur, phosphorus and silicon. [00170] In some embodiments, an amino acid residue has the structure of −N(R^a1)−L^a1−C(R^a2)(R^a3)−L^a2−COO− or a salt form thereof. [00171] In some embodiments, an amino acid analog is a compound in which the amino group and/or carboxylic acid group are independently replaced with an optionally substituted aliphatic or heteroaliphatic moiety. As persons having ordinary skill in the biomedical art know, many amino acid analogs, which mimics structures, properties and/or functions of amino acids, are described in the biomedical art and can be used in accordance with this specification, e.g., in several moieties. In some embodiments, one or more peptide groups are optionally and independently replaced with non-peptide groups. In some embodiments, an amino acid moiety in a polypeptide or peptide is replaced with an amino acid analog moiety. [00172] In some embodiments, moieties are optionally connected to each other through linker moieties. In some embodiments, a reactive group, e.g., RG, is connected to a cellular receptor-binding moiety, e.g., TBT, through a linker, e.g., L^RM. In some embodiments, a moiety, e.g., LG, may also comprise one or more linkers, e.g., L^LG1, L^LG2, L^LG3, L^LG4, etc., to link several portions. In some embodiments, L^LG is a linker moiety described in this specification. In some embodiments, L^LG1 is a linker moiety described in this specification. In some embodiments, L^LG2 is a linker moiety described in this specification. In some embodiments, L^LG3 is a linker moiety described in this specification. In some embodiments, L^LG4 is a linker moiety described in this specification. In some embodiments, L^RM is a linker moiety described in this specification. In some embodiments, L^PM is L. In some embodiments, L^PM is a linker moiety described in this specification. In some embodiments, L^PM is L. [00173] Linker moieties of several types and/or for several purposes, e.g., those used in antibody- drug conjugates, etc., may be used in accordance with this specification. [00174] Linker moieties can be either bivalent or polyvalent depending on how they are used. In some embodiments, a linker moiety is bivalent. In some embodiments, a linker is polyvalent and connecting more than two moieties. [00175] In some embodiments, a linker moiety, e.g., L^z (wherein z represents superscript text; e.g., L^PM, L^RM, L^LG, L^LG1, etc. , is or comprises L. [00176] In some embodiments, L is a covalent bond, or a bivalent or polyvalent optionally substituted, linear or branched C_1-100 group comprising one or more aliphatic, aryl, heteroaliphatic having 1-20 heteroatoms, heteroaromatic having 1-20 heteroatoms, or any combinations thereof, wherein one or more methylene units of the group are optionally and independently replaced with C_1-6

38 30123-WO-PCT alkylene, C_1-6 alkenylene, a bivalent C_1-6 heteroaliphatic group having 1-5 heteroatoms, , −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’)−, an amino acid residue, or −[(−O−C(R’)₂−C(R’)₂−)_n]−, wherein n is 1-20. The linker optionally has a cyclic group, Cy, defined below, and a reactive group, RG as defined below. In some embodiments, each amino acid residue is independently a residue of an amino acid having the structure of formula LNK101 or a salt thereof. In some embodiments, each amino acid residue independently has the structure of −N(R^a1)−L^a1−C(R^a2)(R^a3)−L^a2−CO− or a salt form thereof. [00177] In some embodiments, L is bivalent. In some embodiments, L is a covalent bond. [00178] In some embodiments, L is a bivalent or optionally substituted, linear or branched group selected from C_1-00 aliphatic and C_1-100 heteroaliphatic having 1-50 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with C_1-6 alkylene, C_1-6 alkenylene, a bivalent C_1-6 heteroaliphatic group having 1-5 heteroatoms, , −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’)−, an amino acid residue or −[(−O−C(R’)₂−C(R’)₂−)_n]−. In some embodiments, L is a bivalent or optionally substituted, linear or branched group selected from C_1-20 aliphatic and C_1-20 heteroaliphatic having 1-10 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with C_1-6 alkylene, C_1-6 alkenylene, a bivalent C_1-6 heteroaliphatic group having 1-5 heteroatoms, , −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’)−, an amino acid residue or −[(−O−C(R’)₂−C(R’)₂−)_n]−. In some embodiments, L is a bivalent or optionally substituted, linear or branched group selected from C_1-20 aliphatic wherein one or more methylene units of the group are optionally and independently replaced with , −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’)−, an amino acid residue or −[(−O−C(R’)2−C(R’)2−)n]−. In some embodiments, L is a bivalent or optionally substituted, linear or branched C_1-20 aliphatic wherein one or

39 30123-WO-PCT more methylene units of the group are optionally and independently replaced with , −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−, an amino acid residue or −[(−O−C(R’)₂−C(R’)₂−)_n]−. In some embodiments, L is a bivalent or optionally substituted, linear or branched group C_1-100 aliphatic wherein one or more methylene units of the group are optionally and independently replaced with , −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’)−, an amino acid residue or −[(−O−C(R’)₂−C(R’)₂−)_n]−. In some embodiments, L is a bivalent or optionally substituted, linear or branched group C_1-50 aliphatic wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted, linear or branched group C_1-40 aliphatic wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted, linear or branched group C_1-20 aliphatic wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted, linear or branched group C_1-10 aliphatic wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted, linear or branched group C_1-100 alkylene wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted, linear or branched group C_1-50 alkylene wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted, linear or branched group C_1-40 alkylene wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted, linear or branched group C_1-20 alkylene wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. In some embodiments, L is a bivalent or optionally substituted, linear or branched group C_1-10 alkylene wherein one or more methylene units of the group are optionally and independently replaced as described in this specification. [00179] In some embodiments, a linker moiety, e.g., L, L^PM, L^RM, etc., comprises an acidic group, e.g., −S(O)2OH.

40 30123-WO-PCT [00180] In some embodiments, L is or comprises −[(−O−C(R’)₂−C(R’)₂−)_n]−. In some embodiments, L is or comprises −[(−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 an antibody-binding moiety at a −CH₂−. In some embodiments, −CH₂−CH₂−O− is bonded to a cellular receptor-binding moiety at a −CH₂−. In some embodiments, L^PM is such L. In some embodiments, L^RM is such L. [00181] In some embodiments, a linker moiety, or L, is or comprises −(CH₂CH₂O)n−, wherein each −CH₂− is independently and optionally substituted and n is 1-20. In some embodiments, a linker moiety, or L, is or comprises −(CH₂)n−O−(CH₂CH₂O)n−(CH₂)n−, wherein each n is independently 1-10, and each −CH₂− is independently and optionally substituted. [00182] In some embodiments, a linker moiety is trivalent or polyvalent. In some embodiments, a linker moiety is L, where L is trivalent or polyvalent. In some embodiments, L is trivalent. In some embodiments, L is −CH₂−N(−CH₂−)−C(O)−. [00183] In some embodiments, a linker moiety, e.g., L, comprises one or more amino acid residues or analogs thereof. [00184] In some embodiments, a linker moiety, e.g., L, L^RM, etc., is or comprises a reactive group as described in this specification. In some embodiments, an agent comprises an antibody-binding moiety and a cellular receptor-binding moiety linked through a linker which is or comprises a reactive group. In some embodiments, a reactive group can react with a lysine residue of an antibody in an aqueous buffer. In some embodiments, a reactive group is or comprises −C(O)−O−. In some embodiments, a reactive group is or comprises −C(O)−O−, wherein −O− is bonded to an optionally substituted aryl group. In some embodiments, a reactive group is or comprises −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, an aryl group has the structure , wherein R^s is halogen, −NO₂, -F, −L−R’, −C(O)−L−R’, −S(O)−L−R’, −S(O)₂−L−R’, or −P(O) embodiments, an aryl group has the structure of , wherein each R^s is independently halogen, −NO₂, -F, −L−R’, −C(O)−L−R’,

41 30123-WO-PCT^{−S(O)−L−R’, −S(O)}_{2−L−R’, or −P(O)(−L−R’)2. In some embodiments, an aryl} embodiments, an aryl group is . In some embodiments, C1 is bound to the −O− of −C(O)−O−. In some embodiments, a cellular receptor-binding moiety is at the side of −C(O)− and an antibody- binding moiety is at the side of −O−. [00185] In some embodiments, a linker moiety, e.g., L, L^RM, etc., comprises a reactive group, wherein upon contact with an antibody, the reactive group reacts with a group of the antibody and conjugates a cellular receptor-binding moiety, or a moiety comprising −(Xaa)y−, to the antibody optionally through a linker. In some embodiments, a reactive group is or comprises , wherein the −C(O)− is connected to a cellular receptor-binding moiety, or a moiety comprising −(Xaa)y−, optionally through a linker. In some embodiments, a reactive group is or comprises , wherein the −C(O)− is connected to a cellular receptor-binding moiety, or a moiety comprising −(Xaa)y−, optionally through a linker and the other end of the reactive group is connected to an antibody-binding moiety. [00186] In some embodiments, L is or comprises a bio-orthogonal or enzymatic reaction product moiety. In some embodiments, L is or comprises an optionally substituted triazole moiety, which is optionally part of a bi- or poly-cyclic 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, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, L is or comprises LPET(G)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. [00187] In some embodiments, a linker moiety, e.g., L, L^RM, etc., does not have a reactive group. In some embodiments, a linker moiety, e.g., L, L^RM, etc., does not have a reactive group that readily reacts with proteins under aqueous conditions with pH about 6-9, e.g., physiological conditions. In some embodiments, a linker moiety, e.g., L, L^RM, etc., does not have a reactive group that readily reacts with natural amino acid residues under aqueous conditions with pH about 6-9, e.g., physiological conditions. [00188] In some embodiments, a linker moiety, e.g., L, L^RM, etc., comprises no −S−, wherein none of the two atoms to which the −S− is bonded to is S. In some embodiments, a linker moiety, e.g., L, L^RM,

42 30123-WO-PCT etc., comprises no −S−S−. In some embodiments, a linker moiety, e.g., L, L^RM, etc., comprises no −S− bonded to a beta carbon of a carbonyl group or a double or triple bond conjugated to a carbonyl group. In some embodiments, a linker moiety, e.g., L, L^RM, etc., . In some embodiments, a linker moiety, e.g., L, L^RM, etc., comprises no [00189] In some embodiments, an agent comprises no groups can release LG except one or more optionally in RG. In some embodiments, an agent comprises no −S−S−, acetal or imine groups. In some embodiments, an agent comprises no −S−S−, acetal or imine groups except that the agent may have −S−S− formed by two amino acid residues. In some embodiments, an agent comprises no −S−S−, acetal or imine groups except that the agent may have −S−S− formed by cysteine residues. In some embodiments, an agent comprises no −S−S−, acetal or imine groups. [00190] In some embodiments, L is a covalent bond. In some embodiments, L is a bivalent optionally substituted, linear or branched C_1-100 aliphatic group wherein one or more methylene units of the group are optionally and independently replaced. In some embodiments, L is a bivalent optionally substituted, linear or branched C_6-100 arylaliphatic group wherein one or more methylene units of the group are optionally and independently replaced. In some embodiments, L is a bivalent optionally substituted, linear or branched C_5-100 heteroarylaliphatic group having 1-20 heteroatoms wherein one or more methylene units of the group are optionally and independently replaced. In some embodiments, L is a bivalent optionally substituted, linear or branched C_1-100 heteroaliphatic group having 1-20 heteroatoms wherein one or more methylene units of the group are optionally and independently replaced. [00191] In some embodiments, a linker moiety, 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, a linker moiety is or comprises −(CH₂CH₂O)_n−, wherein n is as described in this specification. In some embodiments, one or more methylene units of L are independently replaced with −(CH₂CH₂O)_n−. [00192] 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

43 30123-WO-PCT embodiments, n is 17. In some embodiments, n is 18. In some embodiments, n is 19. In some embodiments, n is 20. [00193] In some embodiments, a linker moiety 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 amino acid residues. In some embodiments, one or more methylene units of L are independently replaced with an amino acid residue. In some embodiments, one or more methylene units of L are independently replaced with an amino acid residue, wherein the amino acid residue is of an amino acid of formula LNK101 or a salt thereof. In some embodiments, one or more methylene units of L are independently replaced with an amino acid residue, wherein each amino acid residue independently has the structure of −N(R^a1)−L^a1−C(R^a2)(R^a3)−L^a2−CO− or a salt form thereof. [00194] In some embodiments, a linker moiety comprises one or more moieties, e.g., amino, carbonyl, etc., that can be used for connection with other moieties. In some embodiments, a linker moiety comprises one or more −NR’−, wherein R’ is as described in this specification. In some embodiments, −NR’− improves solubility. In some embodiments, −NR’− serves as connection points to another moiety. In some embodiments, R’ is −H. In some embodiments, one or more methylene units of L are independently replaced with −NR’−, wherein R’ is as described in this specification. [00195] In some embodiments, a linker moiety, e.g., L, comprises a −C(O)− group, which can be used for connections with a moiety. In some embodiments, one or more methylene units of L are independently replaced with −C(O)−. [00196] In some embodiments, a linker moiety, e.g., L, comprises a −NR’− group, which can be used for connections with a moiety. In some embodiments, one or more methylene units of L are independently replaced with −N(R’)−. [00197] In some embodiments, a linker moiety, e.g., L, comprises a −C(O)NR’− group, which can be used for connections with a moiety. In some embodiments, one or more methylene units of L are independently replaced with −C(O)N(R’)−. [00198] In some embodiments, a linker moiety, e.g., L, comprises a −C(R’)₂− group. In some embodiments, one or more methylene units of L are independently replaced with −C(R’)₂−. In some embodiments, −C(R’)₂− is −CHR’−. In some embodiments, R’ is −(CH₂)₂C(O)NH(CH₂)₁₁COOH. In some embodiments, R’ is −(CH₂)₂COOH. In some embodiments, R’ is −COOH. [00199] In some embodiments, a linker moiety is or comprises one or more ring moieties, e.g., one or more methylene units of L are replaced with −Cy−. In some embodiments, a linker moiety, e.g., L, comprises an aryl ring. In some embodiments, a linker moiety, e.g., L, comprises an heteroaryl ring. In

44 30123-WO-PCT some embodiments, a linker moiety, e.g., L, comprises an aliphatic ring. In some embodiments, a linker moiety, e.g., L, comprises an heterocyclyl ring. In some embodiments, a linker moiety, e.g., L, comprises a polycyclic ring. In some embodiments, a ring in a linker moiety, e.g., L, is 3-20-membered. In some embodiments, a ring is 5-membered. In some embodiments, a ring is 6-membered. In some embodiments, a ring in a linker is product of a cycloaddition reaction, e.g., click chemistry, and variants thereof) used to link different moieties. [00200] In some embodiments, a linker moiety, e.g., L) is or comprises . In some embodiments, a methylene unit of L is replaced with . In some embodiments, a methylene unit of L is replaced with −Cy−. In some [00201] In some embodiments, a linker moiety, e.g., L) is or comprises −CO)y−. In some embodiments L is or comprises –[(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-, where n, m, p, are independently chosen at each occurrence from 1-20, from 1-12, or 2-10. In some embodiments each n is 2, m is independently chosen at each occurrence from an integer from 2-10, or in some embodiments m is independently chosen from an integer from 2-6 and Cy is . In some embodiments, a methylene unit of L is replaced with −Cy−. In some embodiments, −Cy− . In some embodiments, −Cy− is . agent, comprises in nts, [00204] In some embodiments, L^RM is a covalent bond. In some embodiments, L^RM is not a covalent bond. In some embodiments, L^RM is or comprises −(CH2CH2O)n−. In some embodiments, L^RM is or comprises −(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 is −(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 is −(CH₂)₂−O−(CH₂CH₂O)n−(CH₂)₂−, wherein n is as described in this specification, and each −CH₂− is independently optionally substituted. In some embodiments, L^RM is −(CH₂)₂−O−(CH₂CH₂O)n−(CH₂)₂−, wherein n is as described in this specification. [00205] In some embodiments, L^PM is a covalent bond. In some embodiments, L^PM is not a covalent bond. In some embodiments, L^PM is or comprises −(CH₂CH₂O)n−. In some embodiments, L^PM is or comprises −(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 is −(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 is −(CH2)2−O−(CH2CH2O)n−(CH2)2−, wherein n is as described in this specification, and each −CH2− is independently optionally substituted. In some embodiments, L^PM is −(CH₂)₂−O−(CH₂CH₂O)n−(CH₂)₂−, wherein n is as described in this specification. [00206] In some embodiments, L^PM, e.g., in a product of a first and a second agents) is or comprises a reaction product moiety formed a first reactive moiety and a second reactive moiety. [00207] In some embodiments, a linker moiety, e.g., L^PM in a product of a first and a second agents is or . In some embodiments, a methylene unit of a linker moiety, e.g., L or a linker moiety that L^RM, L^PM, etc. is replaced with −Cy−. In some embodiments, −Cy− is optionally

46 30123-WO-PCT substituted . In some embodiments, −Cy− is . In some embodiments L is – [(CH₂)nO]mCH₂Cy[(CH₂)nO]m-. In some . Cellular receptor-binding moieties [00208] Several receptor-binding moieties, according to embodiments of present invention, are described in Intl. Pat. Publ. WO2019/199621 published October 17, 2019, Intl. Pat. Publ. WO2019/199634 published October 17, 2019, and Intl. Pat. Publ. WO 2021/072246, each of which is incorporated in this specification in its entirety by reference. [00209] In an embodiment, the cellular receptor-binding moiety may include an asialoglycoprotein receptor (ASGPR) binding group connected through an amine group to the linker moiety. [00210] The amine group may be a primary alkyl amine group or secondary alkyl amine group, each of which is optionally substituted on the amine group with a C₁-C₃ alkyl group. [00211] The cellular receptor-binding moiety may include an ASGPR-binding group according to the chemical structure: ,

47 30123-WO-PCT , wherein X is 1-4 atoms in length and comprises O, S, N(R^N1) or C(R^N1)(R^N1) groups such that: when X is 1 atom in length, X is O, S, N(R^N1) or C(R^N1)(R^N1), when X is 2 atoms in length, no more than 1 atom of X is O, S or N(R^N1), when X is 3 or 4 atoms in length, no more than 2 atoms of X are O, S or N(R^N1); wherein R^N1 is H or a C₁-C₃ alkyl group optionally substituted with from 1-3 halo groups; R₁ and R₃ are each independently: H, -(CH₂)_KOH, -(CH₂)_KOC₁-C₄ alkyl, which is optionally substituted with from 1-3 halo groups, C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo groups, -(CH₂)_K-vinyl, O- (CH₂)_K-vinyl, -(CH₂)_K-alkynyl, -(CH₂)_K-COOH, -(CH₂)_KC(O)O-C₁-C₄ alkyl which is optionally substituted with from 1-3 halo groups, O-C(O)-C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo groups, -C(O)-C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo groups, or R₁ and R₃ are each independently group, which is optionally substituted with up to three halo groups, C₁- which alkyl group is optionally substituted with from one to three halo groups or one or two hydroxyl groups, or O-C₁-C₄ alkyl groups, each of which alkyl groups is optionally substituted with from one to three halo groups or one or two hydroxyl groups; and K is independently an integer of 0 to 4, or R₁ and R₃ are each independently a group according to the chemical structure: , wherein R⁷ is O-C1-C4 alkyl, which is optionally substituted with from 1 to 3 halo groups 1 or 2 hydroxy groups, or R⁷ is a -NR^N3R^N4 group or a; or

48 30123-WO-PCT R₁ and R₃ are each independently a group according to the structure: , , ere r 1-2 hydroxyl groups, or a group according to the structure: H, halo (F, Cl, Br, I), CN, NR^N1R^N2, -(CH₂)_KOH, -(CH₂)_KOC₁-C₄ alkyl, which is optionally substituted with from 1-3 halo groups, C₁-C₃ alkyl, which is optionally substituted with from 1-3 halo groups, -O-C₁-C₃-alkyl, which is optionally substituted with from 1-3 halo groups, -(CH₂)_KCOOH, -(CH₂)_KC(O)O-C₁-C₄ alkyl which is optionally substituted with

51 30123-WO-PCT from 1-3 halo groups, O-C(O)-C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo groups, - C(O)-C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo groups, or group which is optionally substituted one groups or one or groups; K is independently an integer of 0 to 4; K’ is an integer of 1 to 4; R^N3 is H, or a C₁-C₃ alkyl group which is optionally substituted with 1-3 halo groups or 1 or 2 hydroxy groups; and R^N4 is H, a C₁-C₃ alkyl group which is optionally substituted with 1-3 halo groups or 1 or 2 hydroxy groups, or R^N4 is a group, where K is preferably 1; is a linker group which comprises at least one IgG4-binding moiety and links the at least one IgG4-binding moiety to the cellular receptor-binding moiety through the optional linker moiety, or is a linker group which has 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 optional linker moiety; group wherein R^N1 and K are the same as above; substituted with up to 3 halo groups and one or two hydroxyl groups, a -(CH₂)_KCOOH group, a -(CH₂)_KC(O)O-C₁-C₄ alkyl group which is optionally substituted

52 30123-WO-PCT with from 1-3 halo groups, a O-C(O)-C₁-C₄ alkyl group, which is optionally substituted with from 1-3 halo F groups, a -C(O)-C₁-C₄ alkyl group, which is optionally substituted with from 1-3 halo groups, a -(CH₂)_K- NR^N3R^N4 group where R^N3 is H, or a C1-C3 alkyl group which is optionally substituted with 1-3 halo groups or 1 or 2 hydroxy groups; and R^N4 is H, a C₁-C₃ alkyl group which is optionally substituted with 1-3 halo groups or 1 or 2 hydroxy groups, or a group, or group, KOC₁-C₄ alkyl, which is optionally substituted with from groups, substituted with from 1-3 halo groups, -(CH₂)_KCOOH, -(CH₂)_KC(O)O-C₁-C₄ alkyl which is optionally substituted with from 1-3 halo groups, O-C(O)-C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo groups, -C(O)-C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo groups, or R^TA is a C₃-C₁₀ aryl or a three- to ten-membered heteroaryl group containing up to 5 heteroaryl atoms, each of said aryl or heteroaryl groups being optionally substituted with up to three (preferably 1) CN, NR^N1R^N2, -(CH₂)_KOH, -(CH₂)_KOC₁-C₄ alkyl, which is optionally substituted with from 1-3 halo groups, C₁-C₃ alkyl, which is optionally substituted with from 1-3 halo groups or 1 or 2 hydroxy groups, -O-C₁-C₃-alkyl, which is optionally substituted with from 1-3 halo groups, -(CH₂)_KCOOH, - (CH₂)_KC(O)O-C₁-C₄ alkyl which is optionally substituted with from 1-3 halo groups, O-C(O)-C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo groups or -(CH₂)_KC(O)-C₁-C₄ alkyl which is optionally substituted with from 1-3 halo groups, or is optionally substituted with up to three C₁-C₃ alkyl groups which are optionally substituted with up to three halo groups, or R^TA is a group, wherein R^N, R^N1 and R^N2 are each independently H or a C₁-C₃ alkyl group which is optionally substituted with from one to three halo groups or one or two hydroxyl groups and each -(CH₂)_K group is optionally substituted with 1-4, preferably 1 or 2, C₁-C₃ alkyl groups which are optionally substituted with from 1-3 fluoro groups or 1-2 hydroxyl groups; and K is independently 0-4. [00212] The cellular receptor-binding moiety may include an ASGPR-binding group according to the chemical structure: or C(R^N1)(R^N1) groups such that: when X is 1 atom in length, X is O, S, N(R^N1) or C(R^N1)(R^N1), when X is 2 atoms in length, no more than 1 atom of X is O, S or N(R^N1), when X is 3 or 4 atoms in length, no more than 2 atoms of X are O, S or N(R^N1); wherein each R^N1 is independently H or a C₁-C₃ alkyl group optionally substituted with from 1-3 halo groups, preferably F (R^N1 is preferably H or methyl, more often H); R₁ and R₃ are each independently H, -(CH₂)_KOH, -(CH₂)_KOC₁-C₄ alkyl, which is optionally substituted with from 1-3 halo (F, Cl, Br, I, preferably F) groups, C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo (F, Cl, Br, I, preferably F) groups, -(CH₂)_Kvinyl, O-(CH₂)_Kvinyl, -(CH₂)_Kalkynyl, -(CH₂)_KCOOH, -(CH₂)_KC(O)O-C₁-C₄ alkyl which is optionally substituted with from 1-3 halo, preferably F groups, O-C(O)-C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo, preferably F groups, -C(O)-C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo, preferably F groups, or R₁ and R₃ are each independently group, which is optionally substituted with up to three (preferably 1) (preferably F), C₁-C₄ alkyl groups, each of which is

54 30123-WO-PCT optionally substituted with from one to three halo groups, preferably F, or one or two hydroxyl groups, or O-C1-C4 alkyl groups, each of which alkyl groups is optionally substituted with from one to three halo groups, preferably F, or one or two hydroxyl groups, and K is independently 0-4 (0, 1, 2, 3 or 4), or R₁ and R₃ are each independently a group according to the chemical structure: , where R⁷ is O-C₁-C₄ alkyl, which is optionally substituted with from 1 to 3 halo groups, preferably F and 1 or 2 hydroxy groups, or R⁷ is a -NR^N3R^N4 group or a , or R₁ and R₃ are ,

55 30123-WO-PCT , , ,

57 30123-WO-PCT or R₁ and R₃ are each independentl group, where is a C₃-C₈ saturated carbocyclic group; R^C is absent, H, C₁-C₄ alkyl which is optionally substituted with from 1-3 halo (preferably fluoro) groups or 1-2 hydroxyl groups, or a group according to the structure: H, halo (F, Cl, Br, I), CN,^{N1 N2} NR R , -(CH₂)_KOH, -(CH₂)_KOC₁-C₄ alkyl, which is optionally substituted with from 1-3 halo (F, Cl, Br, I, preferably F) groups, C₁-C₃ alkyl, which is optionally substituted with from 1-3 halo (F, Cl, Br, I, preferably F) groups, -O-C₁-C₃-alkyl, which is optionally substituted with from 1-3 halo, preferably F groups, - (CH₂)_KCOOH, -(CH₂)_KC(O)O-C₁-C₄ alkyl which is optionally substituted with from 1-3 halo, preferably F groups, O-C(O)-C1-C4 alkyl, which is optionally substituted with from 1-3 halo, preferably F groups, -C(O)- C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo, preferably F groups, or a 30123- group, alkyl group which is optionally substituted with from one to three halo groups, preferably F, or one or two hydroxyl groups; K is independently 0-4 (0, 1, 2, 3 or 4), preferably 0 or 1; K’ is 1-4, preferably 1; R^N3 is H, or a C₁-C₃ alkyl group which is optionally substituted with 1-3 halo groups, preferably F or 1 or 2 hydroxy groups; and R^N4 is H, a C₁-C₃ alkyl group which is optionally substituted with 1-3 halo groups, preferably F or 1 or 2 hydroxy groups, or R^N4 is a group, where K is preferably 1; is a linker group which comprises at least one IgG4-binding moiety and links the at least one IgG4-binding moiety to the cellular receptor-binding moiety through the optional linker moiety, or is a linker group which has 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 optional linker moiety; group where R^N1 and K are the same as above; substituted with up to 3 halo groups (preferably F) and one or two hydroxyl groups, a -(CH₂)_KCOOH group, a -(CH₂)_KC(O)O-C₁-C₄ alkyl group which is optionally substituted with from 1-3 halo, preferably F groups, O-C(O)-C₁-C₄ alkyl group, which is optionally substituted with from 1-3 halo, preferably F groups, a -C(O)-C₁-C₄ alkyl group, which is optionally substituted with from 1-3 halo, preferably F groups, a -(CH₂)_K-NR^N3R^N4 group where R^N3 is H, or a C₁-C₃ alkyl group which is optionally substituted with 1-3 halo groups, preferably F or 1 or 2 hydroxy groups, or

59 30123-WO-PCT group, CH₂)_KOC₁-C₄ alkyl, which is optionally substituted with from 1-3 halo (F, Cl, Br, I, preferably F) groups, C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo (F, Cl, Br, I, preferably F) groups, -(CH₂)_KCOOH, -(CH₂)_KC(O)O-C₁-C₄ alkyl which is optionally substituted with from 1-3 halo, preferably F groups, O-C(O)-C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo, preferably F groups, -C(O)-C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo, preferably F groups, or R^TA is a C₃-C₁₀ aryl or a three- to ten-membered heteroaryl group containing up to 5 heteroaryl atoms, each of said aryl or heteroaryl groups being optionally substituted with up to three (preferably 1) CN, NR^N1R^N2, -(CH₂)_KOH, -(CH₂)_KOC₁-C₄ alkyl, which is optionally substituted with from 1-3 halo (F, Cl, Br, I, preferably F) groups, C₁-C₃ alkyl, which is optionally substituted with from 1-3 halo (F, Cl, Br, I, preferably F) groups or 1 or 2 hydroxy groups, -O-C₁-C₃-alkyl, which is optionally substituted with from 1-3 halo, preferably F groups, -(CH2)KCOOH, -(CH2)KC(O)O-C1-C4 alkyl which is optionally substituted with from 1-3 halo, preferably F groups, O-C(O)-C₁-C₄ alkyl, which is optionally substituted with from 1-3 halo, preferably F groups or -(CH₂)_KC(O)-C₁-C₄ alkyl which is optionally substituted with from 1-3 halo, preferably F groups, or a group, which is optionally substituted with up to three, preferably substituted with up to three halo (preferably F) groups, or

60 30123-WO-PCT p, C₁-C₃ alkyl group which is optionally substituted with from one to three halo groups, preferably F, or one or two hydroxyl groups and wherein each -(CH₂)_K group is optionally substituted with 1-4, preferably 1 or 2, C₁-C₃ alkyl groups which are optionally substituted with from 1-3 fluoro groups or 1-2 hydroxyl groups; and K is independently 0-4 (0, 1, 2, 3 or 4), preferably 0 or 1, or a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof. [00213] In an embodiment, X is -O-C(R^N1)(R^N1), C(R^N1)(R^N1)-O-, -S-C(R^N1)(R^N1), C(R^N1)(R^N1)-S-, N(R^N1)-C(R^N1)(R^N1), C(R^N1)(R^N1)-N(R^N1) or C(R^N1)(R^N1)-C(R^N1)(R^N1) when X is 2 atoms in length, X is -O-C(R^N1)(R^N1)-C(R^N1)(R^N1), C(R^N1)(R^N1)-O-C(R^N1)(R^N1)-, -O-C(R^N1) (R^N1)-O-, -O-C(R^N1) (R^N1)-S-, -O-C(R^N1) (R^N1)-N(R^N1)-, -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) when X is 3 atoms in length, and X is-O-C(R^N1)(R^N1)-C(R^N1)(R^N1)-C(R^N1)(R^N1), C(R^N1)(R^N1)-O-C(R^N1)(R^N1)-(R^N1)(R^N1)-, -O-C(R^N1)(R^N1)- O-C(R^N1)(R^N1)-, -S-C(R^N1)(R^N1)-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)- (R^N1)(R^N1)-S-C(R^N1)(R^N1)-, -S-C(R^N1)(R^N1)-S-C(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(R^N1)-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) when X is 4 atoms in length, wherein R^N1 is the same as stated above. [00214] In an embodiment, X is OCH₂ or CH₂O and R^N1 is H.

61 30123-WO-PCT [00215] The cellular receptor-binding moiety may have the following structure: ; fluoro) groups a or group from 1-3 fluoro groups); Z_A is -(CH₂)_IM, -O-(CH₂)_IM, S-(CH₂)_IM, NR_M-(CH₂)_IM, C(O)-(CH₂)_IM-, a PEG group containing from 1 to 8 preferably 1-4 ethylene glycol residues or a -C(O)(CH₂)_IMNR_M group (preferably a PEG containing group comprising from 1 to 8 ethylene glycol, preferably 2-4 ethylene glycol residues) where IM and R_M are the same as above; and Z_B is absent, (CH₂)_IM, C(O)-(CH₂)_IM- or C(O)-(CH₂)_IM-NR_M, where IM and R_M are the same as above. [00216] In an embodiment, R^A may be a methyl or ethyl group which is optionally substituted with from 1-3 fluoro groups. [00217] In an embodiment, Z_A may be a PEG group containing from 1 to 4 ethylene glycol residues. [00218] In an embodiment, the methyl or ethyl group may be substituted with from 1-3 fluoro groups. [00219] In an embodiment, the ASGPR-binding group may be N-acetyl-D-galactosamine. [00220] In some embodiments, the cellular receptor-binding moiety can be selected from among the following compounds:

62 30123-WO-PCT TABLE 12 ID NO. Chemical Structure TBT103 TBT104 NH O _OH O OH O O O_{O HN} OH N H_{O O} H O HO O O O O O N H O O HO H O O N N NH H H N O O N N O H N N HO O O N O H H O O N N H O O O_O H^{H N} N H O O O O H_O HN TBT105

63 30123-WO-PCT TABLE 12 ID NO. Chemical Structure TBT106 Pharmaceutical composition; pharmaceutically acceptable excipients [00221] Formulations suitable for parenteral administration, such as by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can have antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. Parenteral administration, oral administration, and intravenous administration are methods of administration. The formulations of compounds can be presented in unit- dose or multi-dose sealed containers, such as ampules and vials. [00222] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include these components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

64 30123-WO-PCT Methods of making the agents. [00223] Agents of this specification may be prepared or isolated by synthetic and/or semi-synthetic methods or recombinant methods in accordance with this specification. In some embodiments, polypeptide agents, e.g., cellular receptor-binding moiety peptide agents, maybe be prepared using biological expression systems. In some embodiments, provided agents are prepared synthetically. In some embodiments, provided agents are prepared using certain technologies described in Intl. Pat. Publ. WO 2019/023501, which is incorporated in this specification in its entirety by reference. [00224] Several technologies, e.g., those for preparing antibody-drug conjugates, may be used in preparation of MATE agents. In many such technologies, conjugation is not selective with respect to amino acid residue sites, and product compositions usually have several different types of agents which may differ from each other regarding number of target-binding moieties conjugated and/or conjugation sites. In some embodiments, the invention provides technologies that can be used for selective conjugation of target-binding moieties at amino acid residue sites. [00225] In some embodiments, the invention provides a method of synthesis, comprising the steps of: contacting a first agent comprising a cellular receptor-binding moiety linked to a first reactive group optionally through a first linker with a second agent comprising an antibody moiety linked to a second reactive group optionally through a second linker, wherein the first reactive group reacts with a second reactive group, and forming a product agent comprising a cellular receptor-binding moiety and an antibody- binding moiety optionally through a linker. [00226] In some embodiments, the invention provides a method of synthesis, comprising the steps of: contacting a first composition comprising a plurality of first agents each independently comprising a cellular receptor-binding moiety linked to a first reactive group optionally through a first linker moiety with a second composition comprising a plurality of second agents each independently comprising an antibody moiety optionally linked to a second reactive group optionally through a second linker moiety, wherein a product composition comprising a plurality of product agents each independently comprising a cellular receptor-binding moiety and an antibody-binding moiety optionally through a linker is formed.

65 30123-WO-PCT [00227] In some embodiments, LG is or comprises an antibody-binding moiety as described in this specification, and a linker which links an antibody-binding moiety and RG. [00228] In some embodiments, L^LG2 is −N(R’)−. In some embodiments, L^LG2 is −N(R)−. In some embodiments, L^LG2 is −NH−. [00229] In some embodiments, L^LG2 is optionally substituted bivalent C_1-6 aliphatic. In some embodiments, L^LG2 is −CH₂−. In some embodiments, L^LG2 is −CH₂NH−. In some embodiments, L^LG2 is −CH₂NH−C(O)−. In some embodiments, L^LG2 is −CH₂NH−C(O)−CH₂−. [00230] In some embodiments, L^LG3 is or comprises an optionally substituted aryl ring. In some embodiments, L^LG3 is or comprises an optionally substituted phenyl ring. In some embodiments, L^LG3 is a phenyl ring substituted with one or more electron-withdrawing groups. As understood by persons having ordinary skill in the biomedical art, several electron-withdrawing groups are known in the biomedical art and may be used in accordance with this specification. In some embodiments, an electron-withdrawing group is halogen. In some embodiments, an electron-withdrawing group is −F. In some embodiments, it is −Cl. In some embodiments, it is −Br. In some embodiments, it is −I. In some embodiments, an electron-withdrawing group comprises an X=Y double bond, wherein X is bonded to the group to 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 independently a heteroatom. 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, an electron-withdrawing group is −C(O)−L−R’. In some embodiments, an 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. [00231] In some embodiments, L^LG3 is −L^LG3a−L^LG3b−, wherein L^LG3a is a covalent bond or −C(O)O−CH₂−, wherein −CH₂− is optionally substituted, and L^LG3b is an optionally substituted aryl ring. In some embodiments, L^LG3a is bonded to L^LG2, and L^LG3b is bonded to L^LG4.

66 30123-WO-PCT [00232] In some embodiments, L^LG3a is a covalent bond. In some embodiments, L^LG3a is −C(O)O−CH₂−, wherein −CH₂− is optionally substituted. In some embodiments, L^LG3a is −C(O)O−CH₂−, wherein −CH₂− is substituted. In some embodiments, L^LG3a is −C(O)O−CH2−, wherein −CH2− is unsubstituted. [00233] In some embodiments, a first group, an antibody-binding moiety, and/or LG is released as part of a compound having the structure of R^LG−L^LG1−L^LG2−H or a salt thereof. [00234] In some embodiments, L^LG4 is a covalent bond. In some embodiments, L^LG4 is not a covalent bond. In some embodiments, L^LG4 is −O−. In some embodiments, L^LG4 is −N(R’)−. In some embodiments, L^LG4 is −NH−. In some embodiments, L^LG4 is −N(CH₃)−. In some embodiments, L^LG4 is −N(R’)−, and L^LG3 is −O−. In some embodiments, R’ is optionally substituted C_1-6 alkyl. In some embodiments, L^LG4 is −S−. [00235] In some embodiments, R^LG is or comprises an antibody-binding moiety. In some embodiments, R^LG is or comprises a protein binding moiety. In some embodiments, R^LG is or comprises an antibody-binding moiety. In some embodiments, R^LG is an antibody-binding moiety. In some embodiments, R^LG is a protein-binding moiety. In some embodiments, R^LG is an antibody-binding moiety. [00236] In some embodiments, R^LG is ABT101, R^c−(Xaa)z−, a nucleic acid moiety, or a small molecule moiety. In some embodiments, R^LG is or comprises ABT101. In some embodiments, R^LG is or comprises Rc−(Xaa)z−. In some embodiments, R^LG is or comprises a small molecule moiety. In some embodiments, R^LG is or comprises a peptide agent. In some embodiments, R^LG is or comprises a nucleic acid agent. In some embodiments, R^LG is or comprises an aptamer agent. In some embodiments, an antibody-binding moiety is or comprises ABT101. In some embodiments, a protein-binding moiety is or comprises ABT101. In some embodiments, an antibody-binding moiety is or comprises ABT101. In some embodiments, an antibody-binding moiety is or comprises Rc−(Xaa)z−. In some embodiments, a protein- binding moiety is or comprises Rc−(Xaa)z−. In some embodiments, an antibody-binding moiety is or comprises Rc−(Xaa)z−. [00237] In some embodiments, target-binding moieties may be conjugated to antibody moieties optionally through linker moieties using technologies described in U.S. Pat. Publ.2020/0190165. [00238] In some embodiments, where a particular protecting group (PG), leaving group (LG), or transformation condition is depicted, persons having ordinary skill in the biomedical art know that other protecting groups, leaving groups, and transformation conditions are also suitable and are contemplated. Such groups and transformations are described in Smith & March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5^th edition (John Wiley & Sons, 2001), Larock, Comprehensive Organic Transformations, 2^nd edition (John Wiley & Sons, 1999), and Greene & Wuts,

67 30123-WO-PCT Protecting Groups in Organic Synthesis, 3^rd edition (John Wiley & Sons, 1999), the entirety of each of which is hereby incorporated in this specification by reference. [00239] In some embodiments, leaving groups include but are not limited to, halogens, e.g. fluoride, chloride, bromide, iodide, sulfonates, e.g. mesylate, tosylate, benzenesulfonate, brosylate, nosylate, triflate), diazonium, and the like. [00240] In some embodiments, an oxygen protecting group includes carbonyl protecting groups, hydroxyl protecting groups, etc. Hydroxyl protecting groups are well known in the biomedical art and include those described in Greene & Wuts, Protecting Groups in Organic Synthesis, 3^rd edition (John Wiley & Sons, 1999), the entirety of which is incorporated in this specification by reference. Examples of suitable hydroxyl protecting groups include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetyl), crotonate, 4-methoxy-crotonate, benzoate, p-benylbenzoate, 2,4,6-trimethylbenzoate, carbonates such as methyl, 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl. Examples of such silyl ethers include trimethylsilyl, triethylsilyl, t- butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and other trialkylsilyl ethers. Alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, allyl, and allyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2- methoxyethoxy)methyl, benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. 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- and 4-picolyl. [00241] Amino protecting groups are well known in the biomedical art and include those described in Greene & Wuts, Protecting Groups in Organic Synthesis, 3^rd edition (John Wiley & Sons, 1999), the entirety of which is incorporated in this specification by reference. Suitable amino protecting groups include, but are not limited to, aralkylamines, carbamates, cyclic imides, allyl amines, amides, and the like. Examples of such groups include t-butyloxycarbonyl (BOC), ethyloxycarbonyl, methyloxycarbonyl, trichloroethyloxycarbonyl, allyloxycarbonyl (Alloc), benzyloxocarbonyl (CBZ), allyl, phthalimide, benzyl (Bn), fluorenylmethylcarbonyl (Fmoc), formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, phenylacetyl, trifluoroacetyl, benzoyl, and the like.

68 30123-WO-PCT [00242] Persons having ordinary skill in the biomedical art know that the provided agents may have one or more stereocenters and may be present as a racemic or diastereomeric mixture. Persons having ordinary skill in the biomedical art know there are many methods known in the biomedical art for the separation of isomers to obtain stereoenriched or stereopure isomers of those compounds, including but not limited to high performance liquid chromatography, chiral high performance liquid chromatography, fractional crystallization of diastereomeric salts, kinetic enzymatic resolution, e.g. by fungal- derived, bacterial- derived, or animal-derived lipases or esterases), and formation of covalent diastereomeric derivatives using an enantioenriched reagent. [00243] Persons having ordinary skill in the biomedical art know that several functional groups present in compounds of this specification such as aliphatic groups, alcohols, carboxylic acids, esters, amides, aldehydes, halogens, and nitriles can be interconverted by techniques well known in the biomedical art 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, 5^th edition (John Wiley & Sons, 2001), the entirety of which is incorporated in this specification by reference. Such interconversions may require one or more of the aforementioned techniques, and certain methods for synthesizing compounds of this specification are described below in the Exemplification. [00244] Persons having ordinary skill in the biomedical art know that reaction partners are generally contacted with each other under conditions and for a time sufficient for production of the desired results, e.g., formation of product agents and compositions thereof to desired extents. Many reaction conditions/reaction times may be assessed and used if they are suitable for desired purposes in accordance with this specification; certain such conditions, reaction times, assessment, etc. are described in this specification. [00245] In some embodiments, an agent formed, e.g., a product MATE agent, has the structure of formula AGN101 or AGN102, or a salt thereof. In some embodiments, a cellular receptor-binding moiety in a product agent, e.g., a MATE agent, is the same as a cellular receptor-binding moiety in a reaction partner, e.g., a first agent comprising a cellular receptor-binding moiety) used to prepare a product agent. In some embodiments, an antibody moiety in a product agent, e.g., a MATE agent, is the same as an antibody moiety in a reaction partner, e.g., a second agent comprising an antibody moiety) used to prepare a product agent. [00246] In some embodiments, linker moieties or a part thereof connected to target-binding moieties and/or antibody moieties may be transferred from reaction partners, e.g., L^RM of formula

69 30123-WO-PCT AGN301 or a salt thereof. In some embodiments, a linker moiety in a product agent (may be called L^PM; e.g., L in formula AGN101 or AGN102, is or comprises a linker moiety in a reaction partner, e.g., one between a reactive group and a cellular receptor-binding moiety, e.g., L^RM. In some embodiments, L^PM is or comprises L^RM. In some embodiments, L^PM is −L^RM−L^RG2−. In some embodiments, L^RG2 is −C(O)−. In some embodiments, L^RG2 is −C(O)−, and is bonded to −NH− of a target agent moiety, e.g., −NH− in a side chain of a lysine residue of a protein moiety, which in some embodiments, is an antibody moiety. [00247] Reaction partners, e.g., compounds of formula AGN301 or salts thereof, rarely contain moieties that can react with reactive groups under conditions under which reactive groups react with target agents. In some embodiments, to the extent that some moieties in reaction partners may react with reactive groups under conditions under which reactive groups react with target agents, reactions between such moieties and reactive groups are significantly slower and/or less efficient compared to reactions between reactive groups and target agents. In some embodiments, reactions between such moieties and reactive groups do not significantly reduce, e.g., no more than about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc. of reduction, efficiencies, yields, rates, and/or conversions, etc., of reactions between reactive groups and target agents. In some embodiments, reactive groups, e.g., ester groups, activated carboxylic acid derivatives, etc. react with amino groups,^{e.g., −NH}_{2 groups, of target agents, e.g., protein agents such as antibody agents. In some embodiments,} reaction partners, e.g., compounds of formula AGN301 or salts thereof, do not have amine groups. 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 amine groups. In some embodiments, they do not have primary amine groups (−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, amine groups, e.g., primary amine groups, are capped, e.g., by introduction of acyl groups, e.g., R−C(O)−, e.g., acetyl to form amide groups) to prevent or reduce undesired reactions. [00248] In some embodiments, reactions are performed in buffer systems. In some embodiments, buffer systems of present disclosure maintain structures and/or functions of target agents, cellular receptor-binding moiety, etc. In some embodiments, a buffer is a phosphate buffer. In some embodiments, a buffer is a PBS buffer. In some embodiments, a buffer is a borate buffer. In some embodiments, buffers of this specification provide and optionally maintain certain pH value or range. In some embodiments, a useful pH is about 7-9, e.g., 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, a pH is 7.4. In some embodiments, a pH is

70 30123-WO-PCT 7.5. In some embodiments, a pH is 7.8. In some embodiments, a pH is 8.0. In some embodiments, a pH is 8.2. In some embodiments, a pH is 8.3. [00249] Provided technologies can provide several advantages. In some embodiments, connection of a cellular receptor-binding moiety in a reaction partner, e.g., a compound comprising a reactive group located between an antibody-binding moiety and a cellular receptor-binding moiety, e.g., a composition of matter of formula AGN301 or a salt thereof to an agent comprising an antibody moiety, e.g., a second agent such as an antibody agent, and release of an antibody-binding moiety in a provided reaction partner can be achieved in one reaction and/or in one pot. Thus, in many embodiments, no separate reactions/steps are performed to remove antibody-binding moieties. Persons having ordinary skill in the biomedical art know that by performing connection of cellular receptor-binding moiety and release of antibody-binding moiety in a single reaction/operation, the provided technologies can avoid separate steps for antibody-binding moiety removal and can improve overall efficiency, e.g., by simplify operations, increasing overall yield, etc., reduce manufacturing cost, improve product purity, e.g., by avoiding exposure to antibody-binding moiety removal conditions, which typically involve one or more of reduction, oxidation, hydrolysis, e.g., of ester groups), etc., conditions and may damage target agent moieties, e.g., for protein agent moieties, protein amino acid residues, overall structures, and/or post- translational modifications, e.g., glycans of antibodies) thereof. In some embodiments, provided technologies can provided improved efficiency, e.g., in terms of reaction rates and/or conversion percentages), increased yield, increased purity/homogeneity, and/or enhanced selectivity, particularly compared to reference technologies wherein a reaction partner containing no antibody-binding moieties is used, without introducing step(s) for antibody-binding moiety removal, e.g., antibody- binding moiety is removed in the same step as cellular receptor-binding moiety conjugation. [00250] In some embodiments, the invention provides products of provided processes, which have low levels of damage to antibody moieties compared to processes comprising steps performed for antibody-binding moiety removal but not for substantial conjugation of moieties of interest, e.g. target- binding moieties. In some embodiments, provided product agent compositions have high homogeneity, e.g., regarding the number of cellular receptor-binding moiety per antibody moiety, and/or positions of amino acid residues in antibody moieties conjugated to moieties of interest) compared to reference product compositions, e.g., those from technologies without using antibody-binding moieties, or using extra step(s) for antibody-binding moiety removal, e.g., not using reaction partners described in this specification which comprise a reactive group located between an antibody-binding moiety and a cellular receptor-binding moiety.

71 30123-WO-PCT [00251] In some embodiments, the invention provides a product agent which is an agent comprising an antibody moiety, a cellular receptor-binding moiety and optionally a linker moiety linking an antibody-binding moiety and a cellular receptor-binding moiety. In some embodiments, the invention provides compositions of such agents. [00252] GN3 sortase reagent. A basic protocol for sortase conjugation including the ASPGR binder for the sortase. C-terminal Sortase Tag (LPETGG) for conjugation (GN3/linker). [00253] GN3-maleimide reagent (maleimide-GN3/linker), with a C-terminal Cys for conjugation. MATES [00254] As used herein, the term “MATE” refers to Multimodal Antibody Therapy Enhancer, a next- generation antibody conjugation technology that enables site-directed pairing with therapeutic monoclonal antibodies (mAbs) or therapeutic immunoglobulin (IG) pooled from donors. Persons having ordinary skill in the biomedical art can use MATES materials and methods as guidance to predictable results when making and using the invention. [00255] This agent may be called a MATE agent or MATE. The MATE agents are described, for example, in Intl. Pat. Publ. WO 2021/102052, the content of which is incorporated in this specification in its entirety by reference. In some embodiments, an agent comprises an antibody moiety, a cellular receptor-binding moiety, and a linker moiety linking an antibody moiety and a cellular receptor-binding moiety. [00256] In one embodiment, the IgG4-binding moiety is a Thermo Scientific® CaptureSelect® Biotin Anti-IgG4 (Human) Conjugate (Catalog number 7102902500 or Catalog Number 7102902100). CaptureSelect® Biotin anti-IgG4 (Hu) Conjugate has a 12 kDa recombinant single domain antibody fragment (VHH affinity ligand) that specifically binds to the Fc part of the human IgG4 subclass. The inventors bound the 12 kDa recombinant antibody fragment by a biotin conjugation. The VHH affinity ligand comprises three complementarity-determining regions (CDRs) that form the antigen-binding domain. The affinity ligand is chemically conjugated to biotin via an appropriate spacer that retains the binding reactivity of the ligand when used in combination with streptavidin-based conjugates or streptavidin pre-coated surfaces.

72 30123-WO-PCT Exemplary procedure for conjugation and reaction kinetics analysis. [00257] Buffer exchange: Five mg protein was buffer-exchanged by Amicon® filters and concentrated to target concentration. [00258] Conjugation: 1.5 mg of concentrated antibody was added to a 1.5 mL tube, reagent (10 mM in dimethyl sulfoxide) was added. The total dimethyl sulfoxide concentration in the reaction mixture was 3.74%. The concentration of the reaction is 10 mg/mL. [00259] Incubation: The tube was allowed to approach ambient temperature and then incubated at 25°C. [00260] Sampling and Analysis: At each of the seven reaction times (zero hours, one hour, two hours, three hours, four hours, five hours, and six hours), 10 μL of sample was quenched with four equivalents propylamine and then frozen at -80°C. After the last sample was quenched and frozen at - 80°C for a few minutes, the samples were thawed one by one and analyzed by intact liquid chromatography-mass spectrometry (LC-MS) to obtain MS-DAR. Size exclusion chromatography- high performance liquid chromatography was also acquired to determine purity. [00261] Using 50 mM HEPES, pH 7.0 as the reaction buffer, the ADC reacting dramatically at the first two hours, and 3x reagent was close to DAR6 in five hours. Measurement by liquid chromatography- mass spectrometry. [00262] Different degrees of aggregation occurred while using 50 mM HEPES, pH 7.0 as the reaction buffer and quenching with four equivalents propylamine. Measurement by size exclusion chromatography.

73 30123-WO-PCT [00263] Conjugation process protocol. Plurality of agents. Second agent. [00264] In another embodiment, the invention provides a composition including the agent and at least one additional agent comprising a moiety capable of binding to the antibody that forms the antibody moiety of the first compound. [00265] In some embodiments, a first composition is a composition comprising a first agent as described in this specification. In some embodiments, second agents independently comprise second reactive groups. In some embodiments, a second composition is a composition comprising a plurality of agents as described in this specification, wherein each cellular receptor-binding moiety is independently a reactive group as described in this specification. In some embodiments, a second composition is an antibody composition, wherein antibodies in the composition are not chemically modified. In some embodiments, a second composition is an IVIG preparation. In some embodiments, a product composition is a composition comprising a plurality of agents as described in this specification, wherein each cellular receptor-binding moiety is independently a cellular receptor-binding moiety as described in this specification. [00266] In some embodiments, a cellular receptor-binding moiety in a product agent is a cellular receptor-binding moiety in a first agent. In some embodiments, an antibody moiety in a product agent is

74 30123-WO-PCT an antibody moiety in a second agent. In some embodiments, a second agent is an antibody agent, e.g., a monoclonal antibody, an antibody in a polyclonal antibody, an antibody in an IVIG preparation, etc. In some embodiments, a second reactive group is a function group of an amino acid residue, e.g., −NH2 of Lys, −SH of Cys, etc. In some embodiments, a second reactive group is −NH2 of a Lys residue, e.g., of a residue selected from K246 and K248 of IgG1 heavy chain amino acid residues corresponding thereto, K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto, and K239 and K241 of an IgG4 heavy chain and amino acid residues corresponding thereto. In some embodiments, the invention provides selective reactions at amino acid residues of antibody moieties. [00267] In some embodiments, a second reactive group is installed to an antibody moiety optionally through a linker. In some embodiments, a second reactive group is installed to an antibody moiety through a linker. In some embodiments, a second reactive group is selectively linked to locations of an antibody moiety, e.g., locations selected from K246 and K248 of IgG1 heavy chain amino acid residues corresponding thereto, K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto, and K239 and K241 of an IgG4 heavy chain and amino acid residues corresponding thereto. In some embodiments, the invention provides selective reactions at amino acid residues of antibody moieties. [00268] In some embodiments, the invention provides agents each independently comprising an antibody-binding moiety that binds to an antibody agent, a reactive group, a cellular receptor-binding moiety, and optionally one or more linker moieties linking such groups/moieties. In some embodiments, such agents are useful as reaction partners, e.g., first agents) for conjugating moieties of interest, e.g., target-binding moieties, reactive groups, e.g., second reactive groups) to agents comprising antibody moieties, e.g., second agents). In some embodiments, the invention provides agents for conjugating moieties of interest to antibody moieties in several agents or antibody agents, e.g., monoclonal antibody agents, polyclonal antibody agents, antibody agents of IVIG preparations, etc. In some embodiments, provided agents each comprise a cellular receptor-binding moiety, a reactive group, an antibody-binding moiety, and optionally one or more linker moieties (linkers) linking such moieties. In some embodiments, an antibody-binding moiety is part of a leaving group released after contacting this agent, e.g., a first agent, with an antibody moiety, e.g., of a second agent, and reacting a reactive group of this agent, e.g., a first reactive group of a first agent, with a reactive group of an antibody moiety, e.g., a second reactive group of a second agent, such as −NH2 of a Lys residue of an antibody protein. In some embodiments, provided technologies can provide improved conjugation efficiency, high selectivity,

75 30123-WO-PCT and/or fewer steps (sometimes, single step) to conjugation product agents. In some embodiments, a provided agent, e.g., a first agent, is a composition of matter of formula AGN301 or a salt thereof: LG−RG−LRM−TBT, [AGN301] or a salt thereof, wherein: LG is a group comprising an antibody-binding moiety; RG is a reactive group; LRM is a linker; and TBT is a cellular receptor-binding moiety. [00269] In some embodiments, the invention provides a composition comprising a plurality of agents, wherein each agent independently comprises: an antibody moiety, a cellular receptor-binding moiety, and optionally a linker moiety linking an antibody-binding moiety and a cellular receptor-binding moiety. [00270] In some embodiments, product agents are MATE agents. In some embodiments, an antibody agent moiety comprises IgG Fc region. In some embodiments, an antibody moiety is connected to a cellular receptor-binding moiety through an amino group optionally through a linker. In some embodiments, it is through a lysine residue wherein the amino group of the side chain is connected to a cellular receptor-binding moiety optionally through a linker, e.g., forming −NH−C(O)− as part of an amide group, a carbamate group, etc. [00271] In some embodiments, selected locations of antibody moieties are used for conjugation. In some embodiments, K246 or K248 of an antibody agent (EU numbering, or corresponding residues) are conjugation locations. In some embodiments, a conjugation location is K246 of heavy chain (unless otherwise specified, locations in this specification include corresponding residues in, e.g., modified sequence, e.g., longer, shorter, rearranged, etc., sequences. In some embodiments, a location is K248 of heavy chain. In some embodiments, a location is K288 or K290 of heavy chain. In some embodiments, a location is K288 of heavy chain. In some embodiments, a location is K290 of heavy chain. In some embodiments, a location is K317. In some embodiments, an antibody moiety is a moiety of an IgG1 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an IgG2 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an IgG4 antibody or a fragment thereof. In some embodiments, a composition comprises a plurality of MATE

76 30123-WO-PCT agents, wherein antibody moieties of the plurality of MATE agents are independently an antibody moiety of an IgG1, IgG2, or IgG4 antibody, or a fragment thereof. [00272] In some embodiments, antibody heavy chains are selectively conjugated/labeled over light chains. [00273] This specification can provide controlled cellular receptor-binding moiety, e.g., cellular receptor-binding moiety/antibody moiety ratios, e.g., for cellular receptor-binding moiety being peptide cellular receptor-binding moiety, peptide cellular receptor-binding moiety/antibody ratio (PAR). In some embodiments, a ratio is about 0.1-10, 0.5-6, etc., e.g., 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, about 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, a ratio is of moieties of interest conjugated to antibody moiety and antibody moieties conjugated to moieties of interest, e.g., when a ratio is in the context of a ratio of an agent. In some embodiments, a ratio is of moieties of interest conjugated to antibody moieties and all antibodies in a composition, e.g., when a ratio is in the context of a ratio of a composition. In some embodiments, a ratio is about 0.1-6. In some embodiments, a ratio is about 0.5-2.5. In some embodiments, a ratio is about 0.5-2. In some embodiments, a ratio is about 1-2. In some embodiments, a ratio is about 1.5-2. In some embodiments, a ratio is about 1.5-2 for IgG1, IgG2, and/or IgG4 antibodies or fragments thereof. In some embodiments, for a composition, e.g., target-binding moieties conjugated to IVIG, a ratio is about 1.5-2.5. In some embodiments, a ratio is about 0.1. In some embodiments, a ratio is about 0.2. In some embodiments, a ratio is about 0.3. In some embodiments, a ratio is about 0.4. In some embodiments, a ratio is about 0.5. In some embodiments, a ratio is about 0.6. In some embodiments, a ratio is about 0.7. In some embodiments, a ratio is about 0.8. In some embodiments, a ratio is about 0.9. In some embodiments, a ratio is about 1. In some embodiments, a ratio is about 1.1. In some embodiments, a ratio is about 1.2. In some embodiments, a ratio is about 1.3. In some embodiments, a ratio is about 1.4. In some embodiments, a ratio is about 1.5. In some embodiments, a ratio is about 1.6. In some embodiments, a ratio is about 1.7. In some embodiments, a ratio is about 1.8. In some embodiments, a ratio is about 1.9. In some embodiments, a ratio is about 2. In some embodiments, a ratio is about 2.1. In some embodiments, a ratio is about 2.2. In some embodiments, a ratio is about 2.3. In some embodiments, a ratio is about 2.4. In some embodiments, a ratio is about 2.5. In some embodiments, a ratio is about 1.8 for a composition wherein antibody moieties of a plurality of agents are those of an IVIG preparation. In some embodiments, a ratio of target-binding moieties and antibody moieties is about 1.5-2 wherein antibody

77 30123-WO-PCT moieties of a plurality of agents are those of IgG1. In some embodiments, a ratio of target-binding moieties and antibody moieties is about 1.5-2 wherein antibody moieties of a plurality of agents are those of IgG2. In some embodiments, a ratio of target-binding moieties and antibody moieties is about 1.5-2.0 wherein antibody moieties of a plurality of agents are those of IgG4. In some embodiments, a ratio is about 1.9-2.0. [00274] In some embodiments, in provided agents, e.g., agents of formula AGN101 or AGN102, or a salt thereof) substantially all conjugation sites of antibody moieties have the same modifications, e.g., all share the same moieties of interest optionally connected through the same linker moieties. In some embodiments, no conjugation sites bear different modifications, e.g., different moieties of interest and/or no moieties of interest and/or different linker moieties. [00275] In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., target-binding moieties, conjugated to antibody moieties of a particular type of antibodies, e.g., IgG1, or fragments thereof are conjugated to one or more particularly sites, typically one or two particularly sites, e.g., K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto. In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., target-binding moieties, conjugated to antibody moieties of IgG2 antibodies or fragments thereof are at K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto. In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., target-binding moieties, conjugated to antibody moieties of IgG2 antibodies or fragments thereof are at K239 and K241 of an IgG4 heavy chain and amino acid residues corresponding thereto. In some embodiments, about 10%-100% of all, or substantially all, moieties of interest, e.g., for a plurality of agents, for a composition, etc. are conjugated to antibody moieties of IgG1, IgG2, and/or IgG4 antibodies, or fragments thereof, e.g., for conjugation products with IgG1 antibodies or fragments thereof (antibody moieties being of IgG1 antibodies or fragments thereof), IgG2 antibodies or fragments thereof (antibody moieties being of IgG2 antibodies or fragments thereof), IgG4 antibodies or fragments thereof (antibody moieties being of IgG4 antibodies or fragments thereof), or for conjugation products with IVIG (when certain provided technologies described in this specification are used, selective conjugation with IgG1, IgG2 and IgG4). In some embodiments, a percentage is about 10% or more. In some embodiments, a percentage is about 20% or more. In some embodiments, a percentage is about 25% or more. In some embodiments, a percentage is about 30% or more. In some embodiments, a percentage is about 40% or more. In some embodiments, a percentage is about 50% or more. In some embodiments, a percentage is about 60% or more. In some embodiments, a percentage is about 65% or more. In some embodiments, a percentage

78 30123-WO-PCT is about 70% or more. In some embodiments, a percentage is about 75% or more. In some embodiments, a percentage is about 80% or more. In some embodiments, a percentage is about 85% or more. In some embodiments, a percentage is about 90% or more. In some embodiments, a percentage is about 95% or more. In some embodiments, a percentage is about 100%. [00276] In some embodiments, a composition comprises a plurality of agents, e.g., MATE agents, agents of formula AGN101 or AGN102, or a salt thereof, each independent comprising a cellular receptor-binding moiety, an antibody moiety, and optionally a linker moiety linking a cellular receptor- binding moiety and an antibody moiety. In some embodiments, substantially all target-binding moieties of a plurality of agents are the same. In some embodiments, substantially all target-binding moieties of a plurality of agents comprise peptide moieties of a common amino acid sequence. In some embodiments, substantially all target-binding moieties of a plurality of agents are peptide moieties of a common amino acid sequence. In some embodiments, substantially all conjugation sites of antibody moieties in a plurality of agents have the same modifications, e.g., all share the same moieties of interest optionally connected through the same linker moieties. In some embodiments, no conjugation sites of a plurality of agents bear different modifications, e.g., different moieties of interest and/or no moieties of interest and/or different linker moieties. In some embodiments, a plurality of agents does not have agents that share the same (or substantially the same) antibody moieties but different modifications, e.g., different moieties of interest and/or no moieties of interest and/or different linker moieties. In some embodiments, agents that share the same (or substantially the same) antibody moieties but different modifications, e.g., different moieties of interest and/or no moieties of interest and/or different linker moieties) are intermediates of multiple-step preparations, e.g., comprising steps for removal of antibody-binding moieties in addition to steps for cellular receptor-binding moiety conjugation) of final product agents. [00277] In some embodiments, an antibody moiety is a moiety of an IgG1 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an IgG2 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an IgG3 antibody or a fragment thereof. In some embodiments, an antibody moiety is a moiety of an IgG4 antibody or a fragment thereof. In some embodiments, about 1-100% of all moieties of interest are at common locations. In some embodiments, a cellular receptor-binding moiety is a cellular receptor-binding moiety as described in this specification. In some embodiments, agents of a plurality are each independently of formula AGN101 or AGN102, or a salt thereof.

79 30123-WO-PCT [00278] In some embodiments, antibody moieties of agents of a plurality comprise a common amino acid sequence. In some embodiments, antibody moieties of agents of a plurality comprise a common amino acid sequence in a Fc region. In some embodiments, antibody moieties of agents of a plurality comprise a common Fc region. In some embodiments, antibody moieties of agents of a plurality can bind a common antigen specifically. In some embodiments, antibody moieties are monoclonal antibody moieties. In some embodiments, antibody moieties are polyclonal antibody moieties. In some embodiments, antibody moieties bind to two or more different antigens. In some embodiments, antibody moieties bind to two or more different proteins. In some embodiments, antibody moieties are IVIG moieties. [00279] In some embodiments, a cellular receptor-binding moiety in an agent of a plurality is a cellular receptor-binding moiety. In some embodiments, each cellular receptor-binding moiety is independently a cellular receptor-binding moiety. In some embodiments, a composition comprises a plurality of agents, antibody moieties of agents of the plurality comprise a common amino acid sequence, and agents of a plurality share a common cellular receptor-binding moiety independently linked to a common amino acid residue in the common amino acid sequence, each independently and optionally through a linker; and wherein about 1%-100% of all agents that comprise an antibody moiety that comprises a common amino acid sequence and a common cellular receptor-binding moiety independently comprise a common cellular receptor-binding moiety linked to a common amino acid residue independently and optionally through a linker. In some embodiments, a composition comprises a plurality of agents, antibody moieties of agents of a plurality comprise a common amino acid sequence, and agents of a plurality share a common cellular receptor-binding moiety independently linked to a common amino acid residue in the common amino acid sequence, each independently through a common linker; and wherein about 1%-100% of all agents that comprise an antibody moiety that comprises a common amino acid sequence and a common cellular receptor-binding moiety independently comprise a common cellular receptor-binding moiety linked to a common amino acid residue independently and through a common linker. In some embodiments, a composition comprises a plurality of agents, antibody moieties of agents of a plurality comprise a common amino acid sequence, and agents of a plurality share a common cellular receptor-binding moiety independently linked to a common amino acid residue in the common amino acid sequence, each independently and optionally through a linker; and wherein about 1%-100% of all agents that comprise an antibody moiety that comprises a common amino acid sequence and a common cellular receptor-binding moiety are agents of a plurality. In some embodiments, a composition comprises a plurality of agents, wherein antibody

80 30123-WO-PCT moieties of agents of a plurality comprise a common amino acid sequence, and agents of a plurality share a common cellular receptor-binding moiety independently linked to a common amino acid residue in the common amino acid sequence, each independently through a common linker; and wherein about 1%-100% of all agents that comprise an antibody moiety that comprises a common amino acid sequence, a common cellular receptor-binding moiety, and a common linker are agents of a plurality. [00280] 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. [00281] In some embodiments, a common amino acid sequence comprises 1-1000, 1-500, 1-400, 1- 300, 1-200, 1-100, 1-50, 10-1000, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 20-1000, 20-500, 20- 400, 20-300, 20-200, 20-100, 20-50, 50-1000, 50-500, 50-400, 50-300, 50-200, 50-100, 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, a length is at least 5 amino acid residues. In some embodiments, a length is at least 10 amino acid residues. In some embodiments, a length is at least 50 amino acid residues. In some embodiments, a length is at least 100 amino acid residues. In some embodiments, a length is at least 150 amino acid residues. In some embodiments, a length is at least 200 amino acid residues. In some embodiments, a length is at least 300 amino acid residues. In some embodiments, a length is at least 400 amino acid residues. In some embodiments, a length is at least 500 amino acid residues. In some embodiments, a length is at least 600 amino acid residues. [00282] In some embodiments, a common amino acid sequence is at least 10%-100%, 50%-100%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of an amino acid sequence of an antibody moiety, a protein agent moiety, etc. In some embodiments, it is 10% or more. In some embodiments, it is 20% or more. In some embodiments, it is 30% or more. In some embodiments, it is 40% or more. In some embodiments, it is 50% or more. In some embodiments, it is 60% or more. In some embodiments, it is 70% or more. In some embodiments, it is 80% or more. In some embodiments, it is 90% or more. In some embodiments, it is 100%.

81 30123-WO-PCT [00283] In some embodiments, in a common amino acid sequence, one and only one amino acid residue is linked to a common cellular receptor-binding moiety, e.g., a common cellular receptor-binding moiety. In some embodiments, in a common amino acid sequence, two and only two amino acid residues are linked to a common cellular receptor-binding moiety, e.g., a common cellular receptor- binding moiety. In some embodiments, in a common amino acid sequence, two or more amino acid residues are linked to a common cellular receptor-binding moiety, e.g., a common cellular receptor- binding moiety. In some embodiments, each common cellular receptor-binding moiety, e.g., a common cellular receptor-binding moiety, is independently linked to an amino acid residue in a common amino acid sequence. [00284] In some embodiments, a common amino acid sequence comprises one or more amino acid residues selected from K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto, K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto, and K239 and K241 of an IgG4 heavy chain and amino acid residues corresponding thereto. In some embodiments, a common amino acid sequence comprises one or more amino acid residues selected from K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto. In some embodiments, a common amino acid sequence comprises one or more amino acid residues selected from K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto. In some embodiments, a common amino acid sequence comprises one or more amino acid residues selected from K239 and K241 of an IgG4 heavy chain and amino acid residues corresponding thereto. In some embodiments, a cellular receptor-binding moiety is connected to this amino acid residue (unless explicitly noted, optionally through a linker moiety). In some embodiments, each cellular receptor- binding moiety is connected to this amino acid residue each optionally and independently through a linker moiety. [00285] In some embodiments, antibody moieties share a high percentage of amino acid sequence homology. In some embodiments, the amino acid sequence homology is about 50%-100%. In some embodiments, the amino acid sequence homology is a percentage selected from the Markush group of percentages 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%. [00286] In some embodiments, a percentage used in this specification is a percentage selected from the Markush group of percentages consisting of about 1%-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

82 30123-WO-PCT 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%. [00287] In some embodiments, antibody moiety of agents of a plurality comprises a common Fc region or a fragment thereof. [00288] In some embodiments, moieties of interest of agents of a plurality are at particular locations. In some embodiments, all moieties of interest are at amino acid residues of a common amino acid sequence. In some embodiments, all moieties of interest are at common locations of amino acid residues of a common amino acid sequence. In some embodiments, the number of common locations is 1. In some embodiments, it is 2. In some embodiments, it is 3. In some embodiments, it is 4. In some embodiments, antibody moieties comprise two heavy chains or fragments thereof, and the number of common locations is 2 (one on each chain). In some embodiments, common locations are selected from K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto, K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto, and K239 and K241 of an IgG4 heavy chain and amino acid residues corresponding thereto. [00289] In some embodiments, agents of a plurality share a common cellular receptor-binding moiety independently at least one location. In some embodiments, agents of a plurality share a common cellular receptor-binding moiety and linker independently at least one location. In some embodiments, moieties of interest at two or more or all locations comprise a common cellular receptor-binding moiety. In some embodiments, moieties of interest are the same. [00290] In some embodiments, agents share a common modification at least one common amino acid residue. In some embodiments, agents of a plurality share a common modification at each location connected to a cellular receptor-binding moiety and optionally a linker. In some embodiments, agents of a plurality the same −L^PM−TBT at each location connected to a linker moiety. [00291] In some embodiments, a location is selected from K246, K248, K288, K290, K317 of antibody agents and locations corresponding thereto. In some embodiments, a location is selected from K246 and K248, and locations corresponding thereto. In some embodiments, a location is selected from K288 and K290, and locations corresponding thereto. In some embodiments, a location is K246 or a location corresponding thereto. In some embodiments, a location is K248 or a location corresponding thereto. In some embodiments, a location is K288 or a location corresponding thereto. In some embodiments, a location is K290 or a location corresponding thereto. In some embodiments, a location is K317 or a location corresponding thereto. In some embodiments, a location is K185 of light chain or a location corresponding thereto. In some embodiments, a location is K187 of light chain or a location

83 30123-WO-PCT corresponding thereto. In some embodiments, a location is K133 of heavy chain or a location corresponding thereto. In some embodiments, a location is K246 or K248 of heavy chain or a location corresponding thereto. In some embodiments, a location is K414 of heavy chain or a location corresponding thereto. In some embodiments, a common sequence is a sequence that is about or at least about 10-100, 20-50, e.g., about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, amino acid residues in length, and comprises one or more of such residues or residues corresponding thereto. In some embodiments, a common sequence is a sequence that is about or at least about 10-100, 20-50, e.g., about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, amino acid residues in length, and comprises one, two or more residues selected from K246 and K248 of an IgG1 heavy chain and amino acid residues corresponding thereto, K251 and K253 of an IgG2 heavy chain and amino acid residues corresponding thereto, and K239 and K241 of an IgG4 heavy chain and amino acid residues corresponding thereto. [00292] In some embodiments, about 1%-100% of all agents that comprise an antibody moiety and a cellular receptor-binding moiety are agents of a plurality. In some embodiments, about 1%-100% of all agents that comprise an antibody moiety that comprises a common amino acid sequence and a cellular receptor-binding moiety are agents of a plurality. In some embodiments, about 1%-100% of all agents that comprise an antibody moiety that comprise a common amino acid sequence or can bind to a common antigen and a cellular receptor-binding moiety are agents of a plurality. In some embodiments, about 1%-100% of all agents that comprise an antibody moiety are agents of a plurality. In some embodiments, about 1%-100% of all agents that comprise an antibody moiety that comprise the common amino acid sequence are agents of a plurality. In some embodiments, about 1%-100% of all agents that comprise a protein agent moiety that comprise the common amino acid sequence are agents of a plurality. In some embodiments, a range of all agent agents that comprise an antibody agent moiety that comprise the common amino acid sequence or can bind to the common antigen are selected from a Markush group of ranges where the agents are of a plurality consisting of about 1%- 100%, about 5%-100%, 10%-100%, about 20%-100%, about 25%-100%, about 30%-100%, and about 40%-100%, about 50%-100%. In some embodiments, a range of all agent agents that comprise an antibody agent moiety that comprise the common amino acid sequence or can bind to the common antigen are selected from a Markush group of ranges where the agents are of a plurality consisting of 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, a range of all agent agents

84 30123-WO-PCT that comprise an antibody agent moiety that comprise the common amino acid sequence or can bind to the common antigen are selected from a Markush group of ranges where the agents are of a plurality consisting of 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%. [00293] In some embodiments, each agent of the plurality does not have −S−Cy−, wherein −Cy− is optionally substituted 5-membered monocyclic ring, does not have −S−S− which is not formed by cysteine residues and does not have −SH or salt form thereof that is not of a cysteine residue. In some embodiments, each agent of the plurality does not have −S−CH₂−CH₂−. In some embodiments, each agent of the plurality does not have a moiety that can specifically bind to an antibody agent. In some embodiments, a composition is substantially free from a moiety that can specifically bind to an antibody agent. [00294] In some embodiments, provided agents, compounds, etc., e.g., those of formula AGN301, AGN101, AGN102, etc. and salts thereof have high purity. In some embodiments, a percentage is about 5%-100%. In some embodiments, a percentage is about 10%-100%. In some embodiments, a percentage is about 20%-100%. In some embodiments, a percentage is about 25%-100%. In some embodiments, a percentage is about 30%-100%. In some embodiments, a percentage is about 40%-100%. In some embodiments, a 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

85 30123-WO-PCT 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%. [00295] In some embodiments, the invention provides product agent compositions comprising product agents, e.g., agents of formula AGN101 or AGN102, or a salt thereof. In some embodiments, a product agent composition, e.g., aa agent composition formed from certain methods, comprises a product agent comprising an antibody moiety and a cellular receptor-binding moiety and optionally a linker, e.g., an agent of formula AGN101 or AGN102, or a salt thereof, a released antibody-binding moiety, e.g., a compound comprising R^LG−(L^LG1)_0-1−(L^LG2)_0-1−(L^LG3)_0-1−(L^LG4)_0-1−) or a compound comprising a released antibody-binding moiety, e.g., a compound having the structure of R^LG−(L^LG1)_0-1−(L^LG2)_0-1−(L^LG3)_0-1−(L^LG4)_0-1−H or a salt thereof, and a reaction partner, e.g., a composition of matter of formula AGN301 or a salt thereof. In some embodiments, released antibody-binding moieties may bind to antibody moieties in target agents and/or formed product agents. Several technologies are available to separate released antibody-binding moieties from antibody moieties in accordance with this specification, for example, in some embodiments, contacting a composition with a composition comprising glycine at certain pH. In some embodiments, each agent of a plurality is independently this product agent. Reactive Group [00296] In some embodiments, provided agents, compounds, e.g., those useful as reaction partners such as first agents, comprise reactive groups, e.g., RG. In some embodiments, reactive groups, e.g., RG) are located between antibody-binding moieties, e.g., ABT) and moieties of interest, e.g., MOI), and are optionally and independently linked to antibody-binding moieties and moieties of interest via linkers. In some embodiments, RG is a reaction group as described in this specification. [00297] In some embodiments, reactive groups when used in agents that comprise no antibody- binding moieties react slowly and provide low level of, in some embodiments, substantially no conjugation of moieties of interest with target agents. As demonstrated in this specification, combination of reactive groups with antibody-binding moieties in the same agents, e.g., as in compounds of formula AGN301 or salts thereof, can promote reactions between reactive groups and

86 30123-WO-PCT target agents, enhance reaction efficiency, reduce side reactions, and/or improve reaction selectivity, e.g., in terms of target sites wherein conjugation of moieties of interest with target agents occurs. [00298] Reactive groups in agents can react with several types of groups in target agents. In some embodiments, reactive groups in agents selectively react with amino groups of target agents, e.g., −NH₂ groups on side chains of lysine residues of proteins. In some embodiments, reactive groups when used in agents, e.g., those of formula AGN301 or salts thereof, selectively react with particular sites of target agents, e.g., as shown in examples in this specification, one or more of K246, K248, K288, K290, K317, etc. of IgG1, K251, K 253, etc. for IgG2, K239, K241 for IgG4, etc. In some embodiments, a site is K246 or K248 of an antibody heavy chain. In some embodiments, sites are K246 and/or K248 of an antibody heavy chain. In some embodiments, a site is K246 of an antibody heavy chain. In some embodiments, a site is K248 of an antibody heavy chain. In some embodiments, a site is K288 or K290 of an antibody heavy chain. In some embodiments, a site is K288 of an antibody heavy chain. In some embodiments, a site is K290 of an antibody heavy chain. In some embodiments, a site is K317. In some embodiments, a site is K414 of an antibody heavy chain. In some embodiments, a site is K185 of an antibody light chain. In some embodiments, a site is K187 of an antibody light chain. In some embodiments, sites are K251 and/or K253 of an IgG2 heavy chain. In some embodiments, a site is K251 of an IgG2 heavy chain. In some embodiments, a site is K253 of an IgG2 heavy chain. In some embodiments, sites are K239 and/or K241 of an IgG4 heavy chain. In some embodiments, a site is K239 of an IgG4 heavy chain. In some embodiments, a site is K241 of an IgG4 heavy chain. In some embodiments, conjugation selectively occurs at one or more heavy chain sites over light chain sites. In some embodiments, for technologies without antibody-binding moieties, conjugation occurs at light chain sites more than heavy chain sites. [00299] In some embodiments, a reactive group, e.g., RG, is or comprises an ester group. In some embodiments, a reactive group, e.g., RG, is or comprises an electrophilic group, e.g., a Michael acceptor. [00300] In some embodiments, a reactive group, e.g., RG, is or comprises −L^RG1−L^RG2−, wherein each of L^RG1 and L^RG2 is independently L. In some embodiments, a reactive group, e.g., RG, is or comprises −L^LG4−L^RG1−L^RG2−, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises −L^LG3−L^LG4−L^RG1−L^RG2−, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises −L^LG2−L^LG3−L^LG4−L^RG1−L^RG2−, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises −L^LG4−L^RG2−, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises −L^LG3−L^LG4−L^RG2−, wherein each variable is as described in this specification. In some embodiments, a

87 30123-WO-PCT reactive group, e.g., RG, is or comprises −L^LG2−L^LG3−L^LG4−L^RG2−, wherein each variable is as described in this specification. [00301] In some embodiments, L^LG4 is −O−. In some embodiments, L^LG4 is −N(R)−. In some embodiments, L^LG4 is −NH−. [00302] In some embodiments, L^LG3 is or comprises an optionally substituted aryl ring. In some embodiments, L^LG3 is or comprises a phenyl ring. In some embodiments, an aryl or phenyl ring is substituted. In some embodiments, a substituent is an electron-withdrawing group as described in this specification, e.g., −NO₂, −F, etc. [00303] In some embodiments, L^RG1 is a covalent bond. In some embodiments, L^RG1 is not a covalent bond. In some embodiments, L^RG1 is −S(O)₂−. [00304] In some embodiments, L^RG2 is −C(O)−. In some embodiments, a reactive group is or comprises −L^LG4−C(O)−, wherein each variable is as described in this specification. In some embodiments, a reactive group is or comprises −L^LG3−L^LG4−C(O)−, wherein each variable is as described in this specification. In some embodiments, a reactive group is or comprises −L^LG2−L^LG3−L^LG4−C(O)−, wherein each variable is as described in this specification. [00305] In some embodiments, L^RG2 is −L^RG3−C(=CR^RG1R^RG2)−CR^RG3R^RG4−, wherein each of R^RG1, R^RG2, R^RG3 and R^RG4 is independently −L−R’, and L^RG3 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, each of R^RG1, R^RG2, R^RG3 and R^RG4 is independently R’. In some embodiments, one or more of R^RG1, R^RG2, R^RG3 and R^RG4 is independently −H. In some embodiments, L^RG3 is −C(O)−. In some embodiments, L^RG3 is −C(O)O−. In some embodiments, −O−, −N(R’)−, etc. of L^RG3 is bonded to L^PM. [00306] In some embodiments, R^RG1 is −H. In some embodiments, R^RG3 is −H. [00307] In some embodiments, L^RG2 is optionally substituted −L^RG3−C(=CHR^RG2)−CHR^RG4−, wherein each variable is as described in this specification. [00308] In some embodiments, R^RG2 and R^RG4 are taken together with their intervening atoms to form an optionally substituted ring as described in this specification. In some embodiments, a formed ring is an optionally substituted 3-10-membered monocyclic or bicyclic ring having 0-5 heteroatoms. In some embodiments, a formed ring is an optionally substituted 3-10-membered cycloaliphatic ring. In some embodiments, a formed ring is selected from the Markush group consisting of optionally substituted cycloaliphatic rings consisting of a 3-8-membered cycloaliphatic ring, a 5-8-membered cycloaliphatic ring., a 5-membered cycloaliphatic ring, a 6-membered cycloaliphatic ring, and a 7- membered cycloaliphatic ring. In some embodiments, a formed ring is substituted. In some

88 30123-WO-PCT embodiments, a formed ring is not substituted. In some embodiments, a formed ring has no additional unsaturation in addition to the double bond in C(=CHR^RG2) or C(=CR^RG1R^RG2). [00309] In some embodiments, −C(=CHR^RG2)−CHR^RG4 or −C(=CR^RG1R^RG2)−CR^RG3R^RG4 is optionally [00310] 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)−. [00311] In some embodiments, −L^LG4−L^RG2− is −N(−)−C(O)−, wherein N is a ring atom of an optionally substituted heteroaryl ring. In some embodiments, −L^LG4−L^RG2− is −N(−)−C(O)−, wherein N is a ring atom of L^LG4 which is or comprises an optionally substituted heteroaryl ring. In some embodiments, −L^LG4−L^RG2− is −N(−)−C(O)−O−, wherein N is a ring atom of L^LG4 which is or comprises an optionally substituted heteroaryl ring. [00312] In some embodiments, L^RG2 is optionally substituted −CH₂−C(O)−, wherein −CH₂− is bonded to an electron-withdrawing group comprising or connected to an antibody-binding moiety. In some embodiments, L^RG2 is optionally substituted −CH₂− bonded to an electron-withdrawing group comprising or connected to an antibody-binding moiety. In some embodiments, L^RG1 is an electron-withdrawing group. In some embodiments, L^RG1 is selected from the Markush group consisting of −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)−. [00313] In some embodiments, L^RG2 is optionally substituted −CH₂−C(O)−, wherein −CH₂− is bonded to a leaving group comprising or connected to an antibody-binding moiety. In some embodiments, L^RG2 is optionally substituted −CH₂− bonded to a leaving group comprising or connected to an antibody-binding moiety. In some embodiments, L^RG1 is selected from the Markush group consisting of −O−C(O)−, −OS(O)₂−, −OP(O(OR)−, −OP(O(SR)−, and −OP(O(N(R)₂)−.

89 30123-WO-PCT [00314] In some embodiments, a reactive group reacts with an amino group of a target agent. In some embodiments, an amino group is −NH₂ of the side chain of a lysine residue. [00315] In some embodiments, a target agent is a protein agent. In some embodiments, a target agent is an antibody agent. In some embodiments, a reactive group reacts with an amino acid residue of this protein or antibody agent. In some embodiments, an amino acid residue is a lysine residue. In some embodiments, a reactive group reacts with −NH₂ of the side chain of a lysine residue. In some embodiments, a reactive group is or comprises −C(O)−O−, it reacts with −NH₂, e.g., of the side chain of a lysine residue), and forms an amide group −C(O)−O− with the −NH₂. [00316] In some embodiments, reactive groups, e.g., a first reactive group, a second reactive group, etc., are located at terminal locations. In some embodiments, agents such as first agents comprise first reactive groups linked to target-binding moieties optionally through linker moieties, and do not have antibody-binding moieties. [00317] In some embodiments, the invention provides methods for preparing a composition comprising a plurality of agents, wherein each agent independently comprises: an antibody moiety, a cellular receptor-binding moiety, and optionally a linker moiety linking an antibody moiety and a cellular receptor-binding moiety; which method comprise: contacting a plurality of agents each of which independently comprises a reactive group with a plurality of antibody agents. [00318] In some embodiments, an agent comprising a reactive group comprises an antibody-binding moiety, a cellular receptor-binding moiety and optionally a linker. In some embodiments, agents comprising a reactive group share the same cellular receptor-binding moiety. In some embodiments, agents comprising a reactive group share the same structure. In some embodiments, antibody molecules are of such structures, properties and/or activities to provide antibody moieties in agents described in this specification. In some embodiments, a plurality of antibody molecules comprises two or more IgG subclasses. In some embodiments, a plurality of antibody molecules comprises IgG1. In some embodiments, a plurality of antibody molecules comprises IgG2. In some embodiments, a plurality of antibody molecules comprises IgG4. In some embodiments, a plurality of antibody molecules comprises IgG1 and IgG2. In some embodiments, a plurality of antibody molecules comprises IgG1, IgG2 and IgG4. In some embodiments, a plurality of antibody molecules comprises IgG1, IgG2, IgG3 and IgG4. In some embodiments, a plurality of antibody molecules is IVIG antibody molecules.

90 30123-WO-PCT [00319] In some embodiments, provided agents comprise a reactive group, . In some embodiments, −C(O)− is connected to a cellular receptor-binding moiety, −(Xaa)y−, optionally through a linker and the other end is connected to an In some reacts with an amino group of another moiety, e.g., an antibody moiety, forming an the moiety and releasing a moiety which is or comprises antibody- binding moiety. In some embodiments, an amino group is −NH₂ of a lysine side chain. In some embodiments, −C(O)− is connected to a cellular receptor-binding moiety, or a moiety comprising −(Xaa)y−, optionally through a linker and the other end is connected to R’ or an optional substituent. In some embodiments, provided agents comprise optionally substituted . Such reactive groups may be useful for conjugation with detection, diagnosis, or therapeutic agents. Persons having ordinary skill in the biomedical art know that a variety of agents, and many technologies, e.g., click chemistry, reactions based on functional groups such as amino groups, e.g., amide formation), hydroxyl groups, carboxyl groups, etc. can be used for conjugation in accordance with this specification. [00320] In some embodiments, antibody-binding moieties bind to Fc regions of antibodies. In some embodiments, reactions occur at residues at Fc regions. In some embodiments, target-binding moieties are conjugated to residues of Fc regions, optionally through linker moieties. In some embodiments, a residue is a Lys residue. In some embodiments, an antibody is or comprises IgG1. In some embodiments, an antibody is or comprises IgG2. In some embodiments, an antibody is or comprises IgG4. In some embodiments, an antibody composition used in a method comprises IgG1 and IgG2. In some embodiments, an antibody composition used in a method comprises IgG1, IgG2 and IgG4. In some embodiments, an antibody composition used in a method comprises IgG1, IgG2, IgG3 and IgG4. [00321] In some embodiments, a product is or comprises IgG1. In some embodiments, a product is or comprises IgG2. In some embodiments, a product is or comprises IgG4. In some embodiments, a product composition comprises IgG1 and IgG2. In some embodiments, a product composition comprises IgG1, IgG2 and IgG4. In some embodiments, a product composition comprises IgG1, IgG2, IgG3 and IgG4. [00322] In some embodiments, provided agents comprising antibody moieties provide one or more or substantially all antibody immune activities, e.g. for recruiting one or more types of immune cells

91 30123-WO-PCT and/or provide short-term and long-term immune activities. In some embodiments, provided agents comprising antibody moieties do not significantly reduce one or more or substantially all relevant antibody immune activities. In some embodiments, provided agents comprising antibody moieties improve one or more or substantially all relevant antibody immune activities, e.g., compared to antibody moieties by themselves. In some embodiments, provided agents provides comparable or better stability compared to antibody moieties by themselves, e.g., residence time in blood. In some embodiments, antibody moieties in provided agents can bind to FcRy of immune cells, e.g., several FcRy of immune effector cells for desired immune activities, typically, at comparable or better levels. In some embodiments, antibody moieties in provided agents have comparable Fab/antigen-binding capabilities. In some embodiments, antibody moieties in provided agents have comparable Fab/antigen-binding capabilities. In some embodiments, antibody moieties in provided agents provide FcRn binding. In some embodiments, antibody moieties in provided agents provide FcRn binding, e.g., for antibody recycle and/or prolonged half-life. In some embodiments, provided technologies are useful for modifying blood- derived IgG products as provided technologies are suitable for and can use all IgG subclasses. [00323] In some embodiments, a provided method comprises one of the steps described below. In some embodiments, reacts with an amino group of a lysine side chain to form an amide bond with an antibody molecule, and releases or a salt form thereof. Methods of assessing the chemical structure and function of the agent. [00324] Protein sequencing. In one method of protein sequencing, eight digestions were prepared using five different enzymes (pepsin, Lys C, trypsin, chymotrypsin, Asp N). The digestions for the sample were processed with disulfide reduction, cysteine blocking, and then enzyme digestion. Digestions were analyzed by LC-MS/MS using a Thermo-Fisher Orbitrap fusion™ mass spectrometer. Peptides were characterized from LC-MS/MS data using de nova peptide sequencing and then assembled into antibody sequences. Affinity chromatography [00325] Affinity capture by MabSelect SuRe™ resin for ABT310. Purify at room temperature. Choose the column based on titer, recommended load density (about 20 mg/ml resin). Equilibrate, five column volumes, with the buffer of 25 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, pH 7.5. Load sample.

92 30123-WO-PCT [00326] EQ wash: Wash with the buffer of 25 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, pH 7.5 for three-five column volumes. [00327] Triton wash: Wash with 25 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, pH7.5, 0.05% Triton100, 0.05% Triton 114 for five-ten column volumes. [00328] EQ wash. Wash with 25 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, pH 7.5 buffer for five-ten column volumes. Elute with elution buffer 0.1 M histidine-acetate. Neutralize immediately with 1 M arginine, 400 mM succinate, pH9.0. [00329] Testing for affinity capture eluates: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE ) non-reducing and reducing, and size exclusion chromatography-high performance liquid chromatography. [00330] Affinity capture by Amsphere™ A3 resin for ABT502 and ABT503. Purify at room temperature. Choose the column based on titer, recommended load density (5-10 mg/ml resin). Equilibrate, five column volumes, with the buffer of 25 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, pH 7.5. Sample Load. [00331] EQ wash: Wash with the buffer of 25 mM Tris-HCl, 150 mM sodium chloride, 5 mM EDTA, pH 7.5 for two-five column volumes. Elute with elution buffer 50 mM sodium citrate, 150 mM sodium chloride, pH3.0. Neutralize immediately with 1 M Tris-HCl, pH 9.0. [00332] Testing for affinity capture eluates: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE ) non-reducing and reducing, and size exclusion chromatography-high performance liquid chromatography. [00333] Results: VHH-Cys (ABT502 and ABT503) and VHH-Fc fusion (ABT310) purification. Mixed culture of cells was collected on the 7^th day or when viability <60%. Cell culture fluid was clarified via centrifugation at 10,000 g for 0.5 h and sterile filtered. ABT310 was purified via affinity capture chromatography by MabSelect SuRe™ resin (Cytiva), and ABT502 and ABT503 via affinity capture chromatography by Amsphere A3 Resin (JSR Life Sciences). MabSelect SuRe™ and Amsphere A3 columns were washed extensively (25 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 5 mM EDTA), and eluted via 100 mM 50 histidine-acetate, and 50 mM sodium citrate, pH 3.0, 150 mM sodium chloride, respectively. MabSelect SuRe™ eluate was neutralized in 1 M arginine, 400 mM succinate, pH 9.0, and Amsphere A3 resin, 1 M Tris-HCl, pH 9.0. Size exclusive chromatography [00334] Purify at room temperature. Choose the column based on protein amount and molecule weight.(Superdex 200 pg for ABT310, Superdex 75 pg for ABT502 and ABT503). Equilibrate with a buffer

93 30123-WO-PCT of 20 mM histidine-acetate, 150 mM sodium chloride, pH 5.5 for one column volume. Sample load. Elution with a buffer of 20 mM histidine-acetate, 150 mM sodium chloride, pH 5.5 for one column volume. Collect target protein with desirable purity. [00335] Testing for size exclusion chromatography eluates: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE ) non-reducing and reducing, and size exclusion chromatography-high performance liquid chromatography. [00336] Results: VHH-Cys (ABT502 and ABT503) and VHH-Fc fusion (ABT310) purification. 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. Fractions were then resolved and pooled for application to cation exchange column Poros® XS (Thermo Fisher), pre- equilibrated with 20 mM sodium acetate, pH 5.5 (ABT310), or 20 mM sodium acetate, pH 5.0 (ABT502 or ABT503). Columns were washed extensively and eluted over fifty columns gradient 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 reducing and non-reducing, endotoxin level testing, and liquid chromatography-mass spectrometry to resolve molecular mass. Cation exchange chromatography [00337] Purify at room temperature. Use Poros® XS column. Choose the column based on protein amount, recommended load density (10-20 mg/ml resin). For ABT310, the cation exchange chromatography pH is 5.5. For ABT502 and ABT503, the cation exchange chromatography pH is 5.0. Dilute the sample below 5 mS/cm with 20 mM sodium acetate, pH 5.5/5.0. Equilibrate, five column volumes, with the buffer of 20 mM sodium acetate, pH 5.5/5.0. Sample Load. [00338] EQ wash. Wash with the buffer of 20 mM sodium acetate, pH 5.5/5.0 for 1-5 column volumes. Elute with gradient 0-100% fifty column volumes 20 mM sodium acetate, 1 M sodium chloride, pH 5.5/5.0 (The scale-up gradient can be adjusted based on the pilot run). Strip with 20 mM sodium acetate, 1 M sodium chloride, pH 5.5/5.0 for one-three column volumes. Equilibrate, one column volume, with the buffer of 20 mM sodium acetate, pH 5.5/5.0. Sanitize with 0.5 M sodium hydroxide for thirty minutes. Equilibrate, three-five column volumes, with the buffer of 20 mM sodium acetate, pH 5.5/5.0. [00339] Testing for cation exchange chromatography eluates: Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE ) non-reducing and reducing, and size exclusion chromatography-high performance liquid chromatography.

94 30123-WO-PCT SEC-HPLC [00340] The inventors performed size exclusion chromatography-high performance liquid chromatography analysis on a Vanquish Flex Duo® liquid chromatography instrument using a TSKgel G3000SWxl stainless steel column (7.8 x 300 mm, particle size 5µm). The mobile phase consists of 50 mM sodium phosphate and 300 mM sodium chloride at pH 6.8. Inject 25 µg of sample per run. Perform isocratic elution for fifteen minutes at a flow rate of 1.0 mL/minute. Monitor protein elution by ultraviolet absorbance at 280 nm. Integrate the peaks corresponding to aggregates, monomers, and low molecular weight species, and calculate the percentage of each species. SDS-PAGE [00341] The inventors performed non-reducing and reducing Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis using precast NuPAGE™ 4-12% bis-Tris Gel from Thermo Scientific. Sample loading buffer (4x lithium dodecyl sulfate) is from Invitrogen. Gel running buffer (20x MES) is from GenScript. Treat non-reducing samples with 30 mM iodoacetamide and heat at 95°C for five minutes before analysis. Treat reducing samples with 50 mM dithiothreitol and heat at 95°C for five minutes before analysis. Carry out electrophoresis at a constant voltage of 180 V for forty minutes. Stain gels using Coomassie blue for thirty minutes and destain with water for one hour. Molecular mass analysis by LC-MS [00342] Conduct liquid chromatography-mass spectrometry analysis using Agilent 6230 AdvanceBio LC/TOF system with a PL1912-1502, PLRP-S 1000Å, 2.1 x 50 mm, 5 µm column (Agilent/ PL1912-1502). [00343] For the non-reduced intact mass analysis, dilute 200 pmol sample by ddH₂O, and subject to LC/MS. For the reduced mass analysis, denature and reduce 100 pmol sample 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℃ for twenty minutes, then subjected to LC/MS. The inventors analyze the mass spectroscopy data using Bioconfirm 10.0 software and identify molecules based on molecular masses. Endotoxin level testing: [00344] Dilute purified proteins based on maximum valid dilution (MVD) in LAL reagent water (Charles River/W110). Load samples into Charles River Laboratories EndoSafe LAL Cartridges (Charles River/ PTS11F) and detect them using Charles River Laboratories Endosafe nexgen-MCS™ instrument.

95 30123-WO-PCT Method of assessing agent for administration [00345] Persons having ordinary skill in the biomedical art can analyze an agent formulation for appearance, color, and achromicity using the criteria described by the United States Pharmacopeia– National Formulary in USP <631> or by the European Pharmacopoeia in Ph. Eur.2.2.2. [00346] Persons having ordinary skill in the biomedical art can analyze an agent formulation for appearance and clarity using the criteria described by the United States Pharmacopeia–National Formulary in USP <1061> or by the European Pharmacopoeia in Ph. Eur.2.2.2. [00347] Persons having ordinary skill in the biomedical art can analyze an agent formulation for pH using the criteria described by the United States Pharmacopeia–National Formulary in USP < ˂791> or by the European Pharmacopoeia in Ph. Eur.2.2.3. A generally accepted criterion is ±0.5 pH of the targeted pH. [00348] Persons having ordinary skill in the biomedical art can analyze the drug product for osmolality using the criteria described by the United States Pharmacopeia–National Formulary in USP <785> or by the European Pharmacopoeia in Ph. Eur.2.2.35. [00349] Persons having ordinary skill in the biomedical art can quantify the concentration of an agent by UV-VIS spectroscopy, e.g., at A280 (280 nm). A generally accepted criterion is ±10% of the targeted concentration. [00350] Persons having ordinary skill in the biomedical art can confirm the Identity of an agent by the charge variant peak retention time or pI as measured by imaged capillary isoelectric focusing (iCIEF), a high-resolution technique that separates proteins into groups based on their isoelectric point (pI). [00351] Persons having ordinary skill in the biomedical art can confirm the purity of the agent by the hydrophobic interaction chromatography-high performance liquid chromatography method. For example, the persons having ordinary skill in the biomedical art can confirm the DAR of the agent. A generally accepted criterion is ±10% of the targeted concentration. [00352] Persons having ordinary skill in the biomedical art can confirm the purity of an agent formulation by the size exclusion chromatography-high performance liquid chromatography method. For example, the persons having ordinary skill in the biomedical art can confirm the percentage of a monomer, high molecular weight, and low molecular weight. A generally accepted criterion is a result of ≥90% or ≤5%. [00353] Persons having ordinary skill in the biomedical art can confirm the purity of an agent formulation by the CE-SDS-NR method. For example, the persons having ordinary skill in the biomedical

96 30123-WO-PCT art can confirm the percentage of a main peak and fragment peaks. A generally accepted criterion is a result of ≥85%. [00354] Persons having ordinary skill in the biomedical art can confirm the purity of an agent formulation by the CE-SDS-R method. For example, the persons having ordinary skill in the biomedical art can confirm the percentage of monomer and Minor species peaks. A generally accepted criterion is a result of ≥85%. [00355] Persons having ordinary skill in the biomedical art can confirm the purity of an agent formulation by the imaged capillary isoelectric focusing method. For example, the persons having ordinary skill in the biomedical art can confirm the percentage of charge variants. [00356] Persons having ordinary skill in the biomedical art can confirm the potency of an agent for binding by the ELISA method. A generally accepted criterion is ± a defined percentage as compared to a reference standard. [00357] Persons having ordinary skill in the biomedical art can detect a process impurity (HCP) in an agent formulation by the ELISA method. A generally accepted criterion is ≤ 100 ng/mg. [00358] Persons having ordinary skill in the biomedical art can detect a process impurity (rProtein A) in an agent formulation by the ELISA method. A generally accepted criterion is ≤ 50 ng/mg. [00359] Persons having ordinary skill in the biomedical art can detect an rDNA impurity in an agent formulation by the reverse transcriptase PCR method. A generally accepted criterion is < 10 ng per dose. [00360] Persons having ordinary skill in the biomedical art can confirm the purity of an agent formulation by the RP-high performance liquid chromatography method. For example, the persons having ordinary skill in the biomedical art can confirm the presence of residual free drug & free protein intermediates. [00361] Persons having ordinary skill in the biomedical art can analyze an agent formulation for the presence of endotoxins using the criteria described by the United States Pharmacopeia–National Formulary in USP <85> or by the European Pharmacopoeia in Ph.2.6.14. A generally accepted criterion is dose dependent based on < 5 EU/kg. [00362] Persons having ordinary skill in the biomedical art can analyze a drug substance for its bioburden using the criteria described by the United States Pharmacopeia–National Formulary in USP <61> or by the European Pharmacopoeia in Ph.2.6.12. A generally accepted criterion is ≤ 1 CFU/10 mL. [00363] Persons having ordinary skill in the biomedical art can analyze a drug product for its sterility using the criteria described by the United States Pharmacopeia–National Formulary in USP <71> or by the European Pharmacopoeia in Ph.2.6.1. A generally accepted criterion is no growth.

97 30123-WO-PCT [00364] Persons having ordinary skill in the biomedical art can analyze a drug product for the presence of excipients using the HPLC-ELSD method For example, the persons having ordinary skill in the biomedical art can measure the percentage of surfactants. A generally accepted criterion is ±50% of the targeted value. [00365] Persons having ordinary skill in the biomedical art can analyze a drug product for the subvisible particles in a container using the criteria described by the United States Pharmacopeia– National Formulary in USP <787> or by the European Pharmacopoeia in Ph.2.6.19. Several accepted criterion are known to persons having ordinary skill in the biomedical art. [00366] Persons having ordinary skill in the biomedical art can analyze a drug product for the extractable volume using the criteria described by the United States Pharmacopeia–National Formulary in USP <697> or by the European Pharmacopoeia in Ph.2.6.17. [00367] Persons having ordinary skill in the biomedical art can analyze a drug product for Container Closure Integrity Test (CCIT), using the criteria described by the United States Pharmacopeia–National Formulary in USP <1207>. A bioburden test is usually performed at the final stability time only. A CCIT test is usually performed at repeated time intervals, e.g., annually. Statistical analyses. [00368] Normal distribution quantitative variables can be expressed as means and SDs and compared by an independent-samples t test, as was done by Zhang et al. (October 2019). For non- normally distributed variables, we used median and interquartile range and analyzed with the Mann- Whitney U test. Categorical data was summarized by percentages. A two-sided p value <0.05 was considered statistically significant. All statistical tests were performed using SPSS version 16.0. [00369] % mol/mol= C(free ^^^^^^^^)/[^^(^^^^^^^^ ^^^^^^^^) +BAR× ^^(^^^^^^^^^^^^^^)] × 100, where: BAR: drug to antibody ratio ^^ (^^^^^^^^ ^^^^^^^^): concentration of free drug in ADC (μM) ^^ (^^^^^^^^^^^^^^): concentration of protein in 1 mg/mL ADC (μM) C (p^^^^^^^^^^^^) = 1/[^^^^(^^^^^^^^^^^^^^)] EXAMPLES [00370] The invention is further illustrated by non-limited EXAMPLES.

98 30123-WO-PCT EXAMPLE 1 Transient expression of IgG4-binding moieties in mammalian cells [00371] The inventors seeded and passaged Chinese Hamster Ovary K1 host cells in advance. On the day of transfection, the inventors centrifuged host cells to specific cell density for transfection, then added DNA and transfection reagent (polyethylenimine) into host cell for transfection. Incubated the transfected culture by shaking at 150 rpm. [00372] Shifted temperature from 36.5^oC to 33^oC at twenty-four hours post transfection. Perform feeding on day 0 and day 4, while harvest cell culture on day 7 or when VIA<60%. [00373] Harvest and Clarification: Subject the cell culture fluid to centrifugation at 10000 g for thirty minutes and then pass through a 0.22 µm sterile filter. [00374] Results: VHH-Cys (ABT502 and ABT503) and VHH-Fc fusion (ABT310) expression. Chinese Hamster Ovary K1 cells were cultured to target density in cell culture medium, and heavy chain and light chain vectors with transfection reagent (polyethylenimine) were transiently co-transfected. Transfected cells were cultured in a shaking flask at 150 rpm in an incubator at 36.5^oC for twenty-four hours post- transfection, followed by incubation at 33^oC for remainder of culture time. Feeding was performed on day 0 and day 4. EXAMPLE 2 Material generation [00375] Determination of assay screening conditions. To optimize ligand concentrations and cell lines for screening, HEK-293T and QT6 cells were transfected with plasmids encoding known ligand targets, Protein A (binds antibody Fc; positive control), or vector alone (pUC used as negative control) in 384- well cell-culture plates. After incubation, four four-fold dilutions of each ligand starting at 20 µg/ml were added in quadruplicate to transfected cells, and bound ligand was detected using a single dilution of a fluorescently-labeled secondary antibody in a high-throughput immunofluorescence flow cytometry assay. [00376] Average mean fluorescence intensity (MFI) values were determined for each test ligand dilution in each cell line using ForeCyt® Software (Intellicyt) and plotted using Excel® (Microsoft). The high background rate for each assayed condition was calculated as the percentage of positive events above a defined fluorescence threshold in cells transfected with negative control. Optimal screening concentrations and cell line for each test ligand were determined by the background signal (MFI), and high background rate in the vector control.

99 30123-WO-PCT [00377] Specific agents. Larger agents. VHH-Fc fusion + ASGPR ligand conjugated to the Fc region via MATE. Advantages of AGT401 include the use the MATE conjugation platform, unconjugated Fc fusion is more stable, with superior depth and rate of reduction versus nanobody (bivalent). Advantages of AGT402 include low immunogenicity with well-established conjugation chemistry. [00378] Specific agents. Smaller agents. VHH + ASGPR ligand conjugated via sortase mediated- ligation or C-terminal cysteine. Advantages of AGT403 and AGT404 include that they are smaller agents. VHH + ASGPR ligand conjugated via sortase mediated-ligation or C-terminal cysteine. [00379] Non-Fc fusion degraders show IgG4-depletion. Fc fusion degrader shows IgG4-depletion at eight hours. Fc fusion degrader shows IgG4-depletion and rebound effect at twenty-four hours. [00380] VHH production has a good titer with Chinese Hamster Ovary as a host 500 mg/L in Chinese Hamster Ovary cell. Doing 500 ml scale is useful because recovery is about 50-60% after purification 500 ml, to get 100 mg protein. The manufacturing risks dimer/aggregation during the purification. TABLE 13 Material generation of non-Fc fusion maleimide-based degraders Molecule Expected yield (mg) ABT501 about 300 ABT502 about 400 ABT503 about 400 ABT504 about 300 TABLE 14 Purification: Single step Ni-affinity (Ni Excel-SEC 75) or Protein A (MSS-SEC200) chromatography Agent Concentration Volume Amount Yield SEC-HPLC Endotoxin (mg/mL) (mL) (mg) (mg/L) Purity (%) (EU/mg) ABT311 (Ni Excel- 6.01 140.77 846.03 1692.06 100.00 <0.0126 SEC 75) ABT312 (Ni Excel- 5.19 142.17 737.86 1475.72 100.00 <0.0118 SEC 75) ABT304 (Ni Excel- 6.30 138.07 869.84 1739.68 100.00 <0.0095 SEC 75) ABT305 (Ni Excel- 5.03 139.98 704.10 1408.20 100.00 <0.0128 SEC 75) ABT306 (Ni Excel- 3.59 78.29 281.06 562.12 99.79 <0.0167 SEC 75)

100 30123-WO-PCT TABLE 14 Purification: Single step Ni-affinity (Ni Excel-SEC 75) or Protein A (MSS-SEC200) chromatography Agent Concentration Volume Amount Yield SEC-HPLC Endotoxin (mg/mL) (mL) (mg) (mg/L) Purity (%) (EU/mg) ABT313 (Ni Excel- 6.45 120.83 779.35 1558.70 100.00 0.0114 SEC 75) ABT310 (MSS- 5.42 90.56 490.84 981.68 100.00 <0.0074 SEC200) Expression (purification volume): 0.5 L transient Chinese Hamster Ovary K1 EXAMPLE 3 Specific mutation strategies to reduce FcγR and C1q binding/effector function, and to enhance FcRn binding/exposure of antibodies. [00381] The inventors mutated VHH affinity ligand variant to insert a LALA mutation using biomedical-art recognized methods first described by the Winter group in the 1990s. In this EXAMPLE, LALA = L234A/L235A. [00382] The inventors mutated VHH affinity ligand variant to insert a LALA mutation using biomedical-art recognized methods first described by the Winter group in the 1990s, then inserted P mutations, such as by the biomedical-art recognized methods introduced by Roche team in 2016, such as the technology for adding P329G and P329G combined 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, Volume 29, Issue 10, October 2016, Pages 457–466. This paper shows that even LALA itself abolishes C1q-binding. P329A alone was tested, and abolishes C1q-binding, and reduces FcgR-binding. This paper did not test P329A/LALA but show that P329G/LALA further reduces FcgR-binding beyond LALA alone. [00383] The inventors also mutated VHH affinity ligand variant in other regions. N297A/Q removes natural N-linked glycosylation site in the hinge region. TABLE 15 Molecular Agents and binding moieties weight kDa ABT304 (SEQ ID NO: 9), VHH Org Sort His 14.7 TABLE 15 Molecular Agents and binding moieties weight kDa ABT310 (SEQ ID NO.31). VHH nanobody, truncated hinge, Fc fusion, 77.3 unconjugated. ABT311 (SEQ ID NO: 32). VHH Org His, unconjugated. 14.2 ABT312 (SEQ ID NO: 33). VHH His, truncated hinge, unconjugated. 13.7 ABT313 (SEQ ID NO: 34), VHH His AviTag 15.7 AGT401. VHH w/o hinge region. βGN3 via maleimide. 19.6 AGT402. VHH-Fc fusion. βGN3 via MATE conjugation. is more stable for 83.8 developability. AGT403. VHH w/hinge region. βGN3 via sortase. 18.8 AGT404. VHH w/o hinge region. βGN3 via sortase. 15.3 AGT501. Non-Fc fusion VHH molecules present lower-molecular weights, 14.5 which are advantageous for subcutaneous formulation. Estimated target about 125 mg dose. AGT502. Non-Fc fusion VHH molecules present lower-molecular weights, 14.6 which are advantageous for subcutaneous formulation. Estimated target about 125 mg dose. EXAMPLE 4 VHH epitope on IgG4-Fc [00384] The VHH epitope on IgG4-Fc binding site on IgG4 was determined to be in two parts. Amino acids 47-57 is DVSQEDPEVQFN (SEQ ID NO: 14). Amino acids 106-114 is SNKGPSSI (SEQ ID NO: 15). For the structure of IgG4, see Davies et al., Molecular Immunology, Volume 62, Issue 1, pages 46-53 (November 2014). The inventors overlaid the structure of hIgG1-Fc/FcγRI complex onto the IgG4-Fc model, using the Fc regions. See Kiyoshi et al., Nature Communications, 6, 6866 (2015). The conclusion was that binding of the VHH domain is likely to compete with binding to FcγRI. IgG4 has little or no affinity to other FcγRs or to C1q. [00385] The inventors also overlaid the structure of hIgG1-FcIII peptide complex onto the IgG4-Fc model, using the Fc regions. See Delano et al., Science, 287, 1279-1283 (2000). The conclusion was that the binding of the VHH domain is not likely to compete with binding to the FcIII peptide, or to FcRn (binds in the same region).

102 30123-WO-PCT EXAMPLE 5 Procedure for preparation of AGN402 [00386] AGN402 (ABT310-TBT105). [00387] BAR2 chemical formula (amino acid): C3570H5554N950O1146S24. [00388] Modification: pE(Q)*2,-K*2,G0F,G1F,BAR2. [00389] using a lysine-specific conjugation. [00390] (1) ABT310 in the original buffer (20 mM histidine-acetate, 150 mM sodium chloride, pH 5.5) was buffer exchanged to 20 mM PB, pH 6.5 by UFDF (Pellicon® 3, 10 kDa, 0.11 m²) for 14 DV. Concentration was tested with 16.47 mg/mL by Nanodrop. [00391] (2) 4000 mg of antibody was transferred to 1L bottle (Corning, 430518) and placed in ice bath; then added with conjugation buffer (20 mM PB, pH 6.5) and 10 mM TBT105 solution (three equivalents). The total dimethyl sulfoxide concentration in the reaction mixture was 3.74%; the concentration of the reaction is 10 mg/mL. [00392] (3) The reaction vessel was allowed to approach ambient temperature and then incubated at 25^oC. The reaction was monitored with liquid chromatography-mass spectrometry and BAR value reached 2.00 after ten hours. Then reaction vessel was placed at 4^oC overnight (seventeen hours). [00393] (4) The crude conjugation was then purified with UFDF (Pellicon® 3, 10 kDa, 2*0.11 m²) with phosphate-buffered saline, pH 7.4 (Gibco, 10010031), 15% dimethyl sulfoxide (Sigma, D4540) for 160

103 30123-WO-PCT DV, followed by phosphate-buffered saline, pH 7.4 for 20 DV. Two batches of products were generated, to afford 3776.88 mg of AGN402 (001) (77.30 mg/mL) and 22.44 mg of AGN402 (002) (11.22 mg/mL). [00394] (5) The purified sample was filtered with 0.22 µm filter. [00395] (6) The 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 levels, and endotoxin levels. All the quality control tests produced acceptable and expected results. The measured mass was 83.9 kDa. [00396] The major conjugation site was also confirmed with K154, identified by peptide mapping (Thermo/Q-Exactive Orbitrap). TABLE 16 Residual reagents Residual ABT (% Residual reagent (% mol/mol Residual payload (% mol/mol mol/mol of mAb) of total reagent) of total payload) AGN402 (001) 3.86 1.97 <0.39 AGN402 (002) 1.86 1.02 <0.39 EXAMPLE 6 Procedure for preparation of AGN508 [00397] AGN508 (ABT501-TBT103). [00398] BAR1 chemical formula (amino acid). C671H1042N193O229S5. [00399] Modification. pE(Q),Cys,BAR1. [00400] AGN508 was prepared using a Michael-addition reaction. [00401] (1) Reduction. To the 1L bottle (Corning, 430518) containing 2000 mg of ABT501 in original buffer (7.26 mg/mL in 20 mM histidine-acetate, 150 mM sodium chloride, pH 5.5) was added with 10% (v/v) of 1 M Tris buffer, pH 7.5 to adjust conjugation buffer pH to 6-7, 5 mM Tris(2-carboxyethyl) phosphine hydrochloridesolution (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 reactions were mixed well and incubated at 22^oC for one hour. [00402] (2) Conjugation. Added TBT103 (10 mM in dimethyl sulfoxide) (three equivalents) into the reactions, mixed well and incubated at 22^oC for another one hour.

104 30123-WO-PCT [00403] (3) The crude conjugation was then purified with UFDF (Pellicon® 3, 3 kDa, 2*0.11 m²) with phosphate-buffered saline, pH 7.4 (Gibco, 10010031) for 70 DV, to afford 1949 mg of AGN508. [00404] (4) The purified sample was filtered with 0.22 µm filter. [00405] (5) The 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 levels, and endotoxin levels. All the quality control tests produced acceptable and expected results. The measured mass was 15.6 kDa. EXAMPLE 7 Procedure for preparation of AGN501 [00406] AGN501 (ABT502-TBT103). [00407] BAR1 chemical formula (amino acid). C624H983N170O217S5. [00408] Modification. pE(Q),Cys,BAR1. [00409] AGN501 was prepared using Michael-addition reaction. [00410] (1) Reduction. To the 1L bottle (Corning, 430518) containing 2000 mg of ABT502 in original buffer (7.65 mg/mL in 20 mM histidine-acetate, 150 mM sodium chloride, pH 5.5) was added with 10% (v/v) of 1 M Tris buffer, 7.5 to adjust conjugation buffer pH to 6-7, 5 mM Tris(2-carboxyethyl)phosphine hydrochloride solution (1.5 equivalents) and 200 mM DTPA solution. The protein concentration in the reaction was 5.15 mg/mL. The DTPA concentration in the reaction was 4 mM. The reactions were mixed well and incubated at 22^oC for one hour. [00411] (2) Conjugation. Added TBT103 (10 mM in dimethyl sulfoxide) (three equivalents) into the reactions, mixed well and incubated at 22^oC for another one hour. [00412] (3) The crude conjugation was then purified with UFDF (Pellicon® 3, 3 kDa, 2*0.11 m²) with phosphate-buffered saline, pH 7.4 (Gibco, 10010031) for 70 DV, to afford 1717.8 mg of AGN501. [00413] (4) The purified sample was filtered with 0.22 µm filter. [00414] (5) The 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 levels, and endotoxin levels. All the quality control tests produced acceptable and expected results. The measured mass was 14.5 kDa.

105 30123-WO-PCT EXAMPLE 8 Procedure for preparation of AGN502 [00415] AGN502 (ABT503-TBT103). [00416] BAR1 chemical formula (amino acid). C627H987N170O219S5. [00417] Modification. pE(Q),Cys,BAR1. [00418] AGN502 was prepared using Michael-addition reaction. [00419] (1) Reduction. To the 1L bottle (Corning, 430518) containing 2000 mg of ABT503 in original buffer (7.41 mg/mL in 20 mM histidine-acetate, 150 mM sodium chloride, pH 5.5) was added with 10% (v/v) of 1 M Tris buffer, 7.5 to adjust conjugation buffer pH to 6-7, 5 mM Tris(2-carboxyethyl)phosphine 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 reactions were mixed well and incubated at 22^oC for one hour. [00420] (2) Conjugation. Added TBT103 (10 mM in dimethyl sulfoxide) (three equivalents) into the reactions, mixed well and incubated at 22^oC for another one hour. [00421] (3) The crude conjugation was then purified with UFDF (Pellicon® 3, 3 kDa, 2*0.11 m²) with phosphate-buffered saline, pH 7.4 (Gibco, 10010031) for 70 DV, to afford 2008 mg of AGN502. [00422] (4) The purified sample was filtered with 0.22 µm filter. [00423] (5) The 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 levels, and endotoxin levels. All the quality control tests produced acceptable and expected results. The measured mass was 14.6 kDa. EXAMPLE 9 Procedure for preparation of AGN509 [00424] AGN509 (ABT504-TBT103) [00425] BAR1 chemical formula (amino acid). C660H1025N188O223S5 [00426] Modification. pE(Q),Cys,BAR1. [00427] AGN509 was prepared using a Michael-addition reaction. [00428] (1) Reduction. To the 1L bottle (Corning, 430518) containing 1900 bmg of ABT504 in original buffer (7.78 mg/mL in 20 mM histidine-acetate, 150 mM sodium chloride, pH 5.5) was added with 10% (v/v) of 1 M Tris buffer, 7.5 to adjust conjugation buffer pH to 6-7, 5 mM Tris(2-carboxyethyl)phosphine

106 30123-WO-PCT 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 reactions were mixed well and incubated at 22^oC for one hour. [00429] (2) Conjugation. Added TBT103 (10 mM in dimethyl sulfoxide) (three equivalents) into the reactions, mixed well and incubated at 22^oC for another one hour. [00430] (3) The crude conjugation was then purified with UFDF (Pellicon® 3, 3 kDa, 2*0.11 m²) with phosphate-buffered saline, pH 7.4 (Gibco, 10010031) for 70 DV, to afford 1896.35 mg of AGN509. [00431] (4) The purified sample was filtered with 0.22 µm filter. [00432] (5) The 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 levels, and endotoxin levels. All the quality control tests produced acceptable and expected results. The measured mass was 15.3 kDa. EXAMPLE 10 Procedure for preparation of AGN401 [00433] AGN401 (ABT306-TBT103). [00434] BAR1 chemical formula (amino acid). C719H1115N202O244S5. [00435] Modification. pE(Q),Cys, O-Glycan, one payload.

107 30123-WO-PCT

s prepared using a Michael-addition reaction. [00437] (1) Reduction. To the 150 mL bottle (Corning, 430281) containing 150 mg of ABT306 in original buffer (3.59 mg/mL in 20 mM histidine-acetate, 150 mM sodium chloride, pH 5.5) was added with 10% (v/v) of 1 M Tris buffer, 7.5 to adjust conjugation buffer pH to 6-7, 5 mM Tris(2- carboxyethyl)phosphine 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 reactions were mixed well and incubated at 22^oC for three hours. [00438] (2) Conjugation. Added TBT103 (10 mM in dimethyl sulfoxide) (three equivalents) into the reactions, mixed well and incubated at 22^oC for another one hour. [00439] (3) The crude conjugation was then purified with Amicon® (Millipore, 10 kDa, 15 mL) with phosphate-buffered saline, pH 7.4 (Gibco, 10010031), to afford 135.24 mg of AGN401. [00440] (4) The purified sample was filtered with 0.22 µm filter. [00441] (5) The 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 levels, and endotoxin levels. All the quality control tests produced acceptable and expected results. The measured mass was 19.6 kDa.

108 30123-WO-PCT EXAMPLE 11 Procedure for preparation of AGN403 [00442] AGN403 (ABT304-TBT106). [00443] BAR1 chemical formula. C676H1068N188O235S6. [00444] Modification. pE(Q), BAR1. [00445] AGN403 was prepared using a sortase-catalyzed ligation. [00446] (1) To the 150 mL bottle (Corning, 430281) containing 300 mg of ABT304 in original buffer (6.30 mg/mL in 20 mM histidine-acetate, 150 mM sodium chloride, pH 5.5) was added with 5.08 mg/mL Sortase A (PT-0008, 0.01 equivalents) solution, conjugation buffer (50 mM HEPES, 150 mM sodium chloride, 10% glycerol (V/V), pH 7.5), TBT106 (40 mM in dimethyl sulfoxide) (eight equivalents) into the reactions, mixed well and incubated at 4^oC for overnight (seventeen hours). The ABT304 concentration in the reaction was 3 mg/mL. [00447] (2) The crude conjugation was purified with AKTA pure 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 is one mL/minute). The conjugate was flowed through during the loading process. The sortase with His-tag and unconjugated peptide with His-tag were eluted by 0.2 M sodium hydroxide. The inventors pooled the flow through together and performed buffer exchange to phosphate-buffered saline, 2 mM EDTA, pH 7.4 with Amicon® (Milipore, 10 kDa, 15 mL) for 30 DV. [00448] (3) The purified sample was filtered with 0.22 µm filter. [00449] (4) The 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 levels, and endotoxin levels. All the quality control tests produced acceptable and expected results. The measured mass was 15.8 kDa. EXAMPLE 12 Procedure for Preparation of AGN404 [00450] AGN404 (ABT305-TBT106). [00451] BAR1 chemical formula. C657H1040N182O229S4. [00452] Modification. pE(Q), BAR1. [00453] AGN404 was prepared using Sortase-catalyzed ligation.

109 30123-WO-PCT [00454] (1) To the 150 mL bottle (Corning, 430281) containing 300 mg of ABT305 in original buffer (5.03 mg/mL in 20 mM histidine-acetate, 150 mM sodium chloride, pH 5.5) was added with 5.08 mg/mL Sortase A (PT-0008, 0.01 equivalents) solution, conjugation buffer (50 mM HEPES, 150 mM sodium chloride, 10% glycerol (V/V), pH 7.5), TBT106 (40 mM in dimethyl sulfoxide) (eight equivalents) into the reactions, mixed well and incubated at 4^oC for overnight (seventeen hours). The ABT305 concentration in the reaction was 3 mg/mL. [00455] (2) The crude conjugation was purified with AKTA pure 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 is 1 mL/minutes). The conjugate was flowed through during the loading process. The sortase with His-tag and unconjugated peptide with His-tag were eluted by 0.2 M sodium hydroxide. Pooled flow through together and performed buffer exchange to phosphate-buffered saline, 2 mM EDTA, pH 7.4 with Amicon® (Milipore, 10 kDa, 15 mL) for 30 DV. [00456] (3) The purified sample was filtered with 0.22 µm filter. [00457] (4) The 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 levels, and endotoxin levels. All the quality control tests produced acceptable and expected results. The measured mass was 15.2 kDa. EXAMPLE 13 Procedure for Preparation of Building block #1 Preparation of Intermediate 2 [00458] A mixture of Reactant 1 (20.0 g, 128 mmol, 1.00 equivalent) in ethanol (400 mL) was added Pd(OH)₂ (8.00 g, 56.9 mmol, 0.44 equivalents) and hydrochloric acid (2 M, 64.4 mL, 1.00 equivalent) under nitrogen gas atmosphere. The mixture was stirred at 25 °C for twenty hours under hydrogen gas atmosphere (50 psi). Thin-layer chromatography (DCM: methanol =10: 1) showed the Reactant 1 was consumed and one main spot (R_f = 0.5) was formed. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was triturated with ethyl acetate (100 mL)

110 30123-WO-PCT at 25^oC for two hours to give Intermediate 2 (18.0 g, 92.0 mmol, 71.3% yield, 100% purity, HCl salt) was obtained as a white solid.¹H 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). Preparation of Intermediate 4 [00459] To a solution of Reactant 3 (40.0 g, 78.6 mmol, 1.00 equivalent) in dimethylformamide (200 mL) was added NMM (15.9 g, 157 mmol, 17.3 mL, 2.00 eq) and Intermediate 2 (13.1 g, 67.2 mmol, 0.85 equivalents, HCl salt). The mixture was stirred at 25°C for two hours. The liquid chromatography-mass spectrometry results showed Intermediate 2 was consumed completely. One main peak (retention time = 0.48 minutes) with desired mass (MS calculated was 552.2, MS observed: [M-tBu + H]⁺ = 497.1) was detected. 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 brine 500 mL, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was triturated 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. Liquid chromatography-mass spectrometry was retention time = 0.48 minutes, MS calculated was 552.2. MS found was [M - tBu+H]⁺ = 497.1. Preparation of Building block #1 mL) was added trifluoroacetic acid (154 g, 1.35 mol, 100 mL, 39.2 equivalents). The mixture was stirred at 25°C for two hours. Liquid chromatography-mass spectrometry results showed Intermediate 4 was consumed completely and one main peak (RT = 0.41 minutes) with desired mass (MS calculated was 496.1, MS observed was [M+ H]⁺ = 497.1) was detected. The mixture solution was concentrated under reduced

111 30123-WO-PCT pressure to give a residue. The residue was purified by reversed-phase HPLC (trifluoroacetic acid condition), to give Building block #1 (9.10 g, 17.9 mmol, 52.1% yield, 97.9% purity) as a white solid. Liquid chromatography-mass spectrometry was retention time = 0.41 minutes, MS calculated was 496.1, MS found was [M +Na]⁺ = 519.1. HPLC was retention time = 2.95 minutes, purity was 97.9%, SFC was EC6536-156-P1C, retention time = 1.58 minutes. Value was 97.8%.¹H 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). EXAMPLE 14 Procedure for preparation of TBT105 Preparation of Intermediate 7

112 30123-WO-PCT

[00461] This chemical compound was synthesized using standard Fmoc chemistry (CTC resin). [00462] (1) Resin preparation. To the vessel containing 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 equivalent) in DCM (50 mL) was added N,N- diisopropylethylamine (4.00 equivalents) dropwise and mixed for two hours with nitrogen gas bubbling at 25°C. Then methanol (22.7 mL) was added and bubbled with nitrogen gas for another thirty minutes. The resin was washed with dimethylformamide (100 mL) * 5, followed by the addition of 20% piperidine in dimethylformamide (100 mL) and bubbled with nitrogen gas for thirty minutes at 25°C for Fmoc

113 30123-WO-PCT deprotection. The mixture was filtered. The resin was washed with dimethylformamide (100 mL) * 5 before proceeding to next step. [00463] (2) Coupling. A solution of Fmoc-Cys(Trt)-OH (17.5 g, 30.0 mmol, 3.00 equivalents), HBTU (10.8 g, 28.5 mmol, 2.85 equivalents) in dimethylformamide (500 mL) was added to the resin with nitrogen gas bubbling. Then N,N-diisopropylethylamine (6.00 equivalents) was added to the mixture dropwise and bubbled with nitrogen gas for thirty minutes at 25°C. The coupling reaction was monitored by ninhydrin test, if it showed colorless, the coupling was completed. The resin was then washed with dimethylformamide (300 mL) * 5. [00464] (3) Deprotection.20% piperidine in dimethylformamide (100 mL) was added to the resin and the mixture was bubbled with nitrogen gas for thirty minutes at 25°C. The resin was then washed with dimethylformamide (100 mL) * 5. [00465] (4) Steps 2 and 3 were repeated for the following amino acids elongation. See numbers 3- 15, TABLE 17. [00466] (5) After all the steps were completed, the resin was washed with dimethylformamide (100 mL) * 5, methanol (100 mL) * 5, then dried under reduced pressure to afford resin-bound peptide Intermediate 5 (CTC resin, 10.0 mmol). TABLE 17 The list of amino acids and the corresponding reagents used on SPPS # Materials Coupling reagents 1 Fmoc-Thr(tBu)-OH (3.00 equivalents) DIEA (4.00 equivalents) 2 Fmoc-Cys(Trt)-OH (3.00 equivalents) HBTU (2.85 equiv.) and DIEA (6.00 equiv.) 3 Fmoc-Trp(Boc)-OH (3.00 equivalents) HBTU (2.85 equiv.) and DIEA (6.00 equiv.) 4 Fmoc-Val-OH (3.00 equivalents) HBTU (2.85 equiv.) and DIEA (6.00 equiv.) 5 Fmoc-Leu-OH (3.00 equivalents) HBTU (2.85 equiv.) and DIEA (6.00 equiv.) 6 Fmoc-Glu(OtBu)-OH (3.00 equivalents) HBTU (2.85 equiv.) and DIEA (6.00 equiv.) 7 Fmoc-Gly-OH (3.00 equivalents) HBTU (2.85 equiv.) and DIEA (6.00 equiv.) 8 Fmoc-Leu-OH (3.00 equivalents) HBTU (2.85 equiv.) and DIEA (6.00 equiv.) 9 Building block #1 (2.00 equivalents) DIC (2.00 equiv.) and HOBt (2.00 equiv.) 10 Fmoc-Trp(Boc)-OH (3.00 equivalents) DIC (3.00 equiv.) and HOBt (3.00 equiv.) 11 Fmoc-Ala-OH (3.00 equivalents) DIC (3.00 equiv.) and HOBt (3.00 equiv.) 12 Fmoc-Cys(Trt)-OH (3.00 equivalents) DIC (3.00 equiv.) and HOBt (3.00 equiv.) 13 Fmoc-Asp(OtBu)-OH (3.00 equivalents) DIC (3.00 equiv.) and HOBt (3.00 equiv.)

114 30123-WO-PCT TABLE 17 The list of amino acids and the corresponding reagents used on SPPS # Materials Coupling reagents 14 Ac Ac₂O:NMM:DMF=10:5:85 15 Boc-PEG₂-OH (6.00 equivalents) DIC:HOBt:DMAP=6:6:6 Peptide cleavage and cyclization. [00467] (1) Cleavage solution (TFA/TIS/water, 95/2.5/2.5, v/v/v, 420 mL) was added to the flask containing the side-chain protected resin-bound peptide (CTC resin, 42 g, 10.0 mmol) at 25^oC and stirred for two hours. [00468] (2) After filtration, the filtrate was collected. [00469] (3) The filtrate was precipitated with cold isopropyl ether (2.5 L). After filtration, the solid was washed with isopropyl ether (2.5 L) twice, and the crude peptide was dried under reduced pressure for two hours to afford Intermediate 6 (16.5 g, crude) as a white solid. [00470] To the mixture of Intermediate 6 (16.5 g, crude) in HOAc/MeCN/water (4/3/3, v/v/v, 8 L) was added 0.1 M I₂/AcOH dropwise until a yellow color persisted, then the mixture was stirred at 25^oC for five minutes. The mixture was quenched by addition of 0.1 M aq. Na₂S₂O₃ dropwise until the yellow color disappeared. After filtration, the filtrate was purified by prep-high performance liquid chromatography (A: 0.075% TFA/water, B: MeCN), followed by lyophilization to afford Intermediate 7 (6 g, 90.0% purity, 32.43% yield) as a white solid. Liquid chromatography-mass spectrometry: retention time = 0.960 minutes, MS calculated: M_av = 1851.01, mass observed: [M + 2H]²⁺ =925.90, [M + H]⁺ =1852.01. Preparation of Intermediate 8 (25 mL) was added a mixture of Target A001 (4.09 g, 1.00 equivalent) and N,N-diisopropylethylamine (1.283 g, 1.307 mL, three equivalents) in 50 mL dimethylformamide at 0°C. The resulting reaction was stirred for five minutes at 0°C. After completion monitored by liquid chromatography-mass spectrometry, the mixture was directly injected into the reverse column, purified by prep-high performance liquid chromatography

115 30123-WO-PCT (A: 0.075% TFA/water, B: MeCN), followed by lyophilization to afford Intermediate 8 (2.9 g, 95.0% purity, 50.7% yield) as colorless oil. L Liquid chromatography-mass spectrometry was retention time = 0.709 minutes, MS calculated: Mav = 1821.81, mass observed: [M+16 + 2H]²⁺ =911.60. Preparation of TBT105 7 (2.53 g, 1.38 mmol, 1.00 equivalent) in dimethylformamide (35 mL) was added N,N- diisopropylethylamine (358.73 mg, 2.77 mmol, 459.92 μL, 2.00 equivalents). The mixture was stirred at 20°C for one hour. After completion monitored by liquid chromatography-mass spectrometry, the mixture was acidified by 1 M hydrochloric acid to pH = 5. The mixture was purified by prep-high performance liquid chromatography (A: 0.075% TFA/water, B: MeCN) directly, followed by lyophilization to afford TBT105 (1.52 g, 96.0% purity, 29.98% yield.) as a white solid. Liquid chromatography-mass spectrometry was retention time = 0.817 minutes, MS calculated: M_av = 3505.75, mass observed: [M + 3H]³⁺ =1169.20.

116 30123-WO-PCT EXAMPLE 15 Procedure for preparation of TBT103 [00473] To a solution of Target A001 (25 g, 15.74 mmol, 1.0 eq) in NMP (125 mL, 5 V) was added 2,6- dimethylpyridine (3.37 g, 3.8 mL, 31.49 mmol, 2.0 equivalents). Then, 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. Dropped the mixture into methyl tert-butyl ether (3 L) and pour out the supernatant and get residue. The residue was purification by prep-high performance liquid chromatography twice (column: Phenomenex luna c18250 mm*100 mm* 15um; mobile phase: [A: water (0.2%FA); B: ACN]; B%: 0.00%-20.00%, 21.00 minutes), followed by lyophilization to afford TBT103 (9.5 g, 98.1% purity, 34% yield) as a white solid. Liquid chromatography-mass spectrometry was retention time = 17.129 minutes, MS calculated: M_av = 1548.59, mass observed: [M + H]⁺ =1549.50.

117 30123-WO-PCT EXAMPLE 16 Procedure for preparation of TBT106 [00475] (1) Resin preparation: Dimethylformamide (100.0 mL) was added to the vessel containing Rink Amide MBHA resin (sub: 0.27 mmol/g, 2.00 mmol, 7.5 g). [00476] (2) De-protection: 20% piperidine/dimethylformamide (200.0 mL) was added and mixed at room temperature (25^oC) for thirty minutes. The de-protection reaction was monitored by ninhydrin test, if it showed blue or other brownish red, the reaction was completed. The resin was then washed with dimethylformamide (200.0 mL) *5. [00477] (3) Coupling: Fmoc-amino acid solution in dimethylformamide (100.0 mL) was added to resin with nitrogen gas bubbling, then the activation buffer was added to the resin with nitrogen gas bubbling

118 30123-WO-PCT at room temperature (25^oC) for one hour. The coupling reaction was monitored by ninhydrin test, if it showed colorless, the coupling was completed. The resin was then washed with dimethylformamide (200.0 mL) *5. [00478] (4) Step 2 to 3 was repeated for all other amino acids: (numbers 1-3 in TABLE 18). At the last step, the resin was washed with methanol (200.0 mL) * 3 and dried by nitrogen gas bubbling for three hours. TABLE 18 The list of amino acids and the corresponding reagents used on SPPS # Materials Coupling reagents 1 Fmoc-Pra-OH (1.50 equivalents) HBTU (1.425 equiv.) and DIEA (3.00 equiv.) 2 Fmoc-G-G-OH (2.00 equivalents) HBTU (1.9 equiv.) and DIEA (4.00 equiv.) 3 Fmoc-G-G-G-OH (2.00 equivalents) HBTU (1.9 equiv.) and DIEA (4.00 equiv.) Peptide cleavage and cyclization. [00479] (1) Cleavage solution (TFA/TIS/water, 95/2.5/2.5, v/v/v, 90 mL) was added to the flask containing the side-chain protected resin-bound peptide (Rink Amide MBHA Resin, 9 g, 2.0 mmol) at 25^oC and stirred for two hours. [00480] (2) After filtration, the filtrate was collected. [00481] (3) The filtrate was precipitated with cold isopropyl ether (450 mL). After filtration, the solid was washed with isopropyl ether (450 mL) twice, and the crude peptide was dried under reduced pressure for two hours to afford Intermediate 1 (1 g, crude) as a white solid. The crude peptide was purified by prep-high performance liquid chromatography (A: 0.1% TFA/water, B: MeCN), followed by lyophilization to afford Intermediate 9 (520 mg, 58.23% yield, 89% purity) as a white solid.

119 30123-WO-PCT Preparation of TBT106 [00482] To a solution of Na ascorbate (399.32 mg, 2.02 mmol, three equivalents) in water (5 mL) was added CuSO₄ (0.4 M, 1.68 mL, 1 eq) dropwise at 25^oC under nitrogen gas. The mixture was turned brown, and then THPTA (291.94 mg, 671.89 μmol, one equivalent) was added to get a clear solution A. To a solution B of Target A043 (1.084 g, 671.892 μmol, one equivalent) and Intermediate 9 (300 mg, 671.892 μmol, one equivalent) in water (10 mL) at 25^oC under nitrogen gas was added a solution A. The mixture was stirred at 25^oC for 0.5 hours. After completion monitored by liquid chromatography-mass spectrometry, the crude solution was purified by prep-high performance liquid chromatography (A: 0.1% TFA/water, B: MeCN) to get a crude product. It was further purified by prep-high performance liquid chromatography (A: 0.5% AcOH/water, B: MeCN ), followed by lyophilization to afford TBT106 (590 mg, 96.5% purity, 36% yield) as a white solid. Liquid chromatography-mass spectrometry was retention time =0.55 minutes, MS calculated: M_av =2010.06, mass observed: [M + H]⁺ =2011.1.

120 30123-WO-PCT EXAMPLE 17 Chemical synthesis. Procedure for Preparation of TBT103 intermediate A001A. Preparation of Intermediate 2 mL) was added 1 (34.2 g, 200 mmol, 1.00 equivalent) in 2-methyltetrahydrofuran (160 mL) at 0°C. The mixture was stirred at 25°C for two hours. Thin-layer chromatography (DCM: methanol = 20: 1, R_f = 0.70) showed the reaction was completed, one major new spot with lower polarity was detected. The reaction mixture was added HCl/EA (1 N, 27.0 mL) and stirred for thirty minutes, and the white precipitate was removed by filtration, the filtrate was concentrated under reduced pressure to afford Intermediate 2 (crude, 105.0 g, 370.6 mmol) as yellow oil. Liquid chromatography-mass spectrometry retention time = 0.797 minutes, MS calculated: 283.14, mass observed: [M + Na]⁺ = 306.1.¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.23-7.41 (m, 5 H), 5.01 (s, 2 H), 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). Preparation of Intermediate 3 (85.6 g, 385 mmol, 1.50 equivalents) and stirred at 60°C for two hours. The reaction was then cooled to room temperature (25°C) and stirred for another one hour. A mixture of Intermediate 2 (80.0 g, 282 mmol, 1.10 equivalents) and 4 Å powder molecular sieves (50.0 g) in DCE (500 mL) was added to the reaction. The resulting mixture was stirred for thirty minutes under N₂ atmosphere. 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 for sixteen hours at 25°C under N2 atmosphere. Thin-layer chromatography

121 30123-WO-PCT (DCM: methanol = 10: 1, R_f = 0.42) indicated Intermediate 2a was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was filtered and washed with sat. 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 a residue. The residue was purified by column chromatography (SiO₂, PE: EA = 3: 1 to 1: 6, then DCM: methanol = 20: 1) to afford Intermediate 3 (90.0 g, 146.9 mmol, 91.6% purity, 57.2% yield) as yellow oil. Liquid chromatography-mass spectrometry was room temperature = 0.860 minutes, MS calculated: 612.25, mass observed: [M + H]⁺ = 613.2.¹H NMR (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). Preparation of Intermediate 4 [00485] Pd/C (9.00 g, 10% purity) in reaction bottle (purged with argon gas for three times) was added tetrahydrofuran (180 mL) slowly, then 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 added to the reaction slowly under nitrogen gas. The reaction was degassed and purged with nitrogen gas and hydrogen gas for three times, then stirred at 25°C for three hours under hydrogen gas atmosphere (40 psi). Thin-layer chromatography (DCM: methanol = 10: 1, R_f = 0.20) indicated Intermediate 3 was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was dissolved in tetrahydrofuran (100 mL), filtered carefully through siliceous earth under nitrogen gas atmosphere, the cake was washed with tetrahydrofuran (100 mL * 2), and the filtrate was concentrated under reduced pressure to get the residue. The residue was diluted with water (1000 mL), washed with DCM (300 mL * 3), the aqueous layer was lyophilized to afford Intermediate 4 (80.0 g, 139.0 mmol, 95.1% purity, 91.8% yield, trifluoroacetic acid salt) as a white solid. Liquid chromatography-mass spectrometry was retention time = 0.484 minutes, MS calculated: 478.22, mass observed: [M + H]⁺ = 478.9.¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.91 (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,

122 30123-WO-PCT 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). Preparation of Intermediate 6 added aqueous sodium hydroxide (5.0 M, 9.91 mL, 0.10 equivalents) dropwise at 0-15°C for over five minutes. After addition, the mixture was stirred at 0-15°C for 5 minutes, then 5a (254.0 g, 1.98 mol, 287 mL, 4.00 equivalents) was added to the reaction mixture dropwise at 20°C. The resulting mixture was stirred at 25°C for sixteen hours. Thin-layer chromatography (DCM: methanol = 10: 1, Rf = 0.7) indicated Intermediate 5 was consumed completely, and one major new spot with lower 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 addition of 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 afford Intermediate 6 (100.0 g, 197.8 mmol, 96.0% purity, 40.0% yield) as colorless oil.¹H 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). Preparation of Intermediate 7 [00487] To a solution of Intermediate 6 (40.0 g, 79.1 mmol, 1.00 equivalent) in MeCN (400 mL) was added HOBt (10.7 g, 79.1 mmol, 1.00 equivalent). 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 was stirred at 25°C for sixteen hours. TLC (PE: EA = 1: 1, R_f = 0.80) indicated Intermediate 6 was consumed completely, and one major new spot with lower polarity was detected. MeCN was evaporated to get the residue. The residue was purified by column chromatography (SiO₂, PE: EA = 10: 1 to 1: 1) to afford Intermediate 7 (40.0 g, 57.4 mmol, 82.9% purity, 72.5% yield) as a white solid. Liquid chromatography-mass spectrometry was retention time = 1.151 minutes, MS calculated: 696.38, mass observed: [M + H]⁺ = 697.3, [M + Na]⁺ = 719.3.¹H NMR (400 MHz, 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, 27 H). Preparation of Intermediate 8 stirred at 25°C for sixteen hours. Thin-layer chromatography (PE: EA = 1: 1, R_f = 0.04) indicated Intermediate 7 was consumed completely, and one major new spot with larger polarity was detected. Solvent was evaporated under reduced pressure, then co-evaporated with toluene (50 mL * 3) under reduced pressure and dried under reduced pressure to get the residue. The residue was purified by prep-high performance liquid chromatography (A: 0.1% FA condition/water, B: MeCN) to afford Intermediate 8 (20.0 g, 37.8 mmol, 98.2% purity, 87.9% yield).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 was retention time = 0.790 minutes, MS calculated: 528.20, mass observed: [M + H]⁺ = 529.2.

124 30123-WO-PCT Preparation of Intermediate 9. [00489] To a stirring solution of Intermediate 8 (20.0 g, 37.8 mmol, 1.00 equivalent) and Intermediate 4 (78.5 g, 132 mmol, 3.50 equivalents, TFA salt) in dimethylformamide (400 mL) was added HOBT (20.4 g, 151 mmol, 4.00 equivalents), EDCI (29.0 g, 151 mmol, 4.00 equivalents) and N,N- diisopropylethylamine (22.0 g, 170 mmol, 4.50 equivalents) successively. The reaction was stirred at 25°C for two hours. Thin-layer chromatography (DCM: methanol = 10: 1, R_f = 0.4) indicated Intermediate 8 was consumed completely, and one major new spot with larger polarity was detected. The reaction mixture was slowly poured into a stirring cold 0.5 mol/L hydrochloric acid solution (900 mL) and stirred for ten minutes. White precipitate was formed and filtered. The aqueous phase was extracted with DCM (600 mL* 2) twice. The combined organic layers were washed with 5% NaHCO₃ (450 mL), dried over Na₂SO₄, and concentrated under reduced pressure to get a residue. The residue was purified by column chromatography (SiO₂, DCM: methanol = 100: 1 to 5: 1) to afford Intermediate 9 (58.0 g, 30.4 mmol, 82.7% purity, 80.3% yield) as a white solid.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, 9 H), 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 was retention time = 3.455 minutes, MS calculated: 1908.81, mass observed: [M + 2H]²⁺ = 955.7.

125 30123-WO-PCT Preparation of Intermediate 10. [00490] The 500 mL round-bottom flask was purged with argon gas for three times and added dry Pd/C (1.50 g, 1.41 mmol, 10% purity, 1.00 equivalent) carefully. Then tetrahydrofuran (150.0 mL) was added to infiltrate the Pd/C completely, followed by the 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) in tetrahydrofuran (75 mL) slowly under argon gas atmosphere. The resulting mixture was degassed and purged with hydrogen gas for three times. Then the mixture was stirred at 25°C for three hours under hydrogen gas atmosphere (15 psi). The reaction was monitored by liquid chromatography-mass spectrometry, which showed the desired mass (one main peak with desired was detected.). The reaction mixture was filtered carefully through siliceous earth under nitrogen gas atmosphere, the cake was washed with tetrahydrofuran (100 mL*2). Then, the filter cake was added water immediately. The organic layer concentrated under reduced pressure to afford Intermediate 10 (13.0 g, 6.54 mmol, 83.2% yield, 99.7% purity, trifluoroacetic acid salt) as a white solid.¹H 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, 3 H), 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 was retention time = 1.333 minutes, MS calculated: 1774.7, found: [M + 2H]²⁺ = 888.6.

126 30123-WO-PCT Preparation of intermediate Target A001A in methanol (120.0 mL) was added NaOMe (5.4 M, 5.01 mL, 4.26 equivalents) at 0°C. The mixture was stirred at 0°C for 0.5 hours. The reaction was monitored by liquid chromatography-mass spectrometry, which showed the desired mass (one main peak with desired mass was detected.). The reaction mixture was added with 1.0 M hydrochloric acid solution (10.0 mL) till the pH = 6. The mixture was diluted with water (75.0 mL) and extracted with DCM (120 mL * 3). The mixture was freeze-dried to afford 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 = 4.0 Hz, 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 was retention time = 0.966 minutes, MS calculated: 1396.6, found: [M + 2H]²⁺ = 699.1. EXAMPLE 18 Chemical synthesis. Procedure for preparation of TBT103 [00492] To a solution of Target A001A (500 mg, 357 μmol, 1.00 equivalent) and 3- maleimidopropionic acid N-hydroxysuccinimide ester (142.869 mg, 536 μmol, 1.50 equivalents) in

127 30123-WO-PCT dimethylformamide (5 mL) was added N,N-diisopropylethylamine (46.2 mg, 357 μmol, 59.1 μL, 1.00 equivalent) degassed and purged with nitrogen gas for three times. The mixture was stirred at 0°C for five minutes under nitrogen gas atmosphere. Liquid chromatography-mass spectrometry showed the desired mass (one main peak with desired was detected.). The reaction mixture was purified by prep- high performance liquid chromatography (AcOH condition) directly to afford TBT103 (251.2 mg, 156 μmol, 43.7% yield, 96.5% purity) as a colorless solid. Liquid chromatography-mass spectrometry was retention time = 1.382 minutes, MS calculated: M_av = 1548.59, [M + H]⁺ = 1548.9, [M + 2H]²⁺ = 775.1. TABLE 19 Assay specification for TBT103 Test Analytical Method Acceptance Criteria Appearance¹ Visual inspection Identification by FTIR, USP <197A> The infrared absorption spectrum of sample conforms FTIR to reference standard. Identification by HPLC The retention time of the main peak of sample HPLC Assay solution conforms to that in the reference standard solution ≥95.0% (On anhydrous basis and solvent-free basis) Related substances HPLC Individual impurity: ≤1.2% Unspecified impurities: ≤5.0% Residual solvents HS-GC W_ater^{KF, USP <921>}_{≤ 10%} EXAMPLE 19 Membrane Proteome Array [00493] Antibody deconvolution and specificity profiling. The Membrane Proteome Array (MPA) is a platform for profiling the specificity of antibodies and other ligands that target human membrane proteins. The MPA was used to determine antibody target specificity and deconvolute orphan antibody/ligand targets. Membrane Proteome Array platform uses flow cytometry to directly detect antibody binding to membrane proteins expressed in unfixed cells. The target proteins have native conformations and appropriate post-translational modifications. [00494] Ligands were tested for reactivity against our library of over 6,000 human membrane proteins, including 94% of all single-pass, multi-pass, and GPI-anchored proteins, including GPCRs, ion channels, and transporters. Identified targets were validated in secondary screens to confirm reactivity.

128 30123-WO-PCT [00495] Materials and methods. Membrane Proteome Array (MPA) screening was conducted at Integral Molecular, Inc. The MPA is a protein library composed of 6,000 distinct human membrane protein clones, each overexpressed in live cells from expression plasmids. Each clone was individually transfected in separate wells of a 384-well plate followed by a twenty-four hour incubation. See Tucker et al., Proc. Natl. Acad. Sci. USA.115,E4990- E4999 (2018). Cells expressing each individual MPA protein clone were arrayed in duplicate in a matrix format for high-throughput screening. Before screening on the Membrane Proteome Array, the test ligand concentration for screening was determined on cells expressing positive (membrane- tethered Protein A) and negative (mock-transfected) binding controls, followed by detection by flow cytometry using a fluorescently-labeled secondary antibody. Each test ligand was added to the MPA at the predetermined concentration, and binding across the protein library was measured on an Intellicyt iQue using a fluorescently labeled secondary antibody. Each array plate contains both positive (Fc- binding) and negative (empty vector) controls to ensure plate-by-plate reproducibility. Test ligand interactions with any targets identified by MPA screening were confirmed in a second flow cytometry experiment using serial dilutions of the test antibody, and the target identity was re-verified by sequencing. [00496] The final screen results are summarized in the following Tables. The project was carried out in three phases: TABLE 20 Membrane Proteome Array carried out in three phases I. Determination of assay screening conditions. Conditions appropriate for detecting test ligand binding by high-throughput flow cytometry were determined, including the optimal ligand concentration and cell type for screening. II. Membrane Proteome Array screen. Binding targets were identified by screening test ligands on the Membrane Proteome Array. III. Validation of ligand targets. Newly identified targets were validated in a follow-up titration experiment using flow cytometry. TABLE 21 Test ligands with newly identified targets, results Test Ligand Screening Cell Screening Known Targets Newly Identified Targets Name Line Concentration, HGNC Uniprot HGNC Uniprot µg/mL AGT402 HEK-293T 20 ASGR1 P07306 GPER1 Q99527 ADRA1B P35368

129 30123-WO-PCT [00497] Optimization of ligand concentrations for screening. Serial dilutions of each test ligand were assayed by incubation with known targets, and target binding was measured by flow cytometry. The optimal ligand concentration for screening was chosen based on a joint assessment of binding strength and background signal (left) and rate of high background events. [00498] Membrane Proteome Array screen. To identify ligand binding targets, about 6,000 different membrane proteins were each expressed in individual wells of HEK-293T cells arrayed in 384-well plates. The cells were then matrixed by pooling individual columns and rows of each plate, so that each protein was represented in a unique combination of two different wells of the matrix plate. [00499] Targets were identified by detecting ligand binding (using previously optimized conditions) to overlapping column and row pools, thereby allowing specific deconvolution. Each individual membrane protein target was assigned values corresponding to the binding values of their unique row and column pools, and targets demonstrating binding signal >3 standard deviations above background in both wells were selected for downstream validation experiments. The resulting paired binding values were subsequently normalized and transformed to give a single numerical value for binding of the test ligand against each target protein. Non-specific fluorescence was determined to be any value below three standard deviations of the mean background value. [00500] Membrane Proteome Array screen results. Each ligand was tested for binding by flow cytometry against the MPA at the previously determined optimal concentration. Binding interactions confirmed in downstream validation studies are displayed in blue and any proteins that did not pass validation were removed. [00501] Target validation. To validate any off-target interaction identified, cells were transfected with plasmids encoding the identified targets, protein A, or vector alone in 384-well format. After incubation four four-fold dilutions of each test ligand, starting at 20 µg/ml, were added to transfected cells followed by detection of ligand binding using a high-throughput immunofluorescence flow cytometry assay. [00502] Average mean fluorescence intensity (MFI) values were determined for each ligand dilution. Only validated targets demonstrating a dose response and mean fluorescence intensity ≥2-fold above background at two consecutive concentrations were tested. [00503] Validation results for newly identified ligand targets. Serial ligand dilutions were incubated with newly identified targets and binding was measured by flow cytometry. A target was validated if it demonstrated binding signal ≥2-fold above background at two consecutive concentrations tested.

130 30123-WO-PCT EXAMPLE 20 Surface plasmon resonance. In vitro evaluation to characterize on-mechanism target-engagement. [00504] Surface plasmon resonance assay. Surface plasmon resonance (SPR) assay is a biophysical approach that provides a label-free and real-time method for measuring biomolecular interactions, including binding affinity, kinetics, and specificity. The assay is performed on a biosensor platform where the binding interaction occurs between a ligand immobilized on a sensor chip and an analyte flowing in solution. Resonance signal (RU) plotted against analyte concentration derives a K_D via a “steady-state” fit. [00505] The use of surface plasmon resonance (Biacore™ and IBIS-MX96 systems) and bio-layer interferometry (ForteBio™ Octet™ systems) can measure the binding ability of the IgG4-binding moiety. The assay can be performed on a Biacore S200 instrument, which has high sensitivity and low-medium throughput, e.g., using the manufacturer’s instructions. The IgG4-binding can be coupled to surface of a sensor chip as the ligand. As analyte is flowed in solution over immobilized ligand, binding to the sensor chip surface induces a change in refractive index proportional to bound mass. IgG4 Fc surface plasmon resonance protocol [00506] Background: The objective of this assay was to determine binding of bifunctional conjugates to IgG4 Fc using surface plasmon resonance. [00507] This protocol was developed to confirm on-target engagement of protein-based degraders and pre-conjugated intermediate (monofunctional binders) with IgG4 Fc protein. The experimental setup uses a Biacore S200 instrument but can be adapted to other Biacore instrumentation such as the 8K or 8K+. [00508] Preparation: Reconstitute lyophilized IgG4 Fc protein in diH₂O according to manufacturer instructions. For 200 µg pack size Human IgG4 Fc protein from ACRO Biosystems, reconstitute lyophilized material with 400 µL diH₂O and gently mix at room temperature for thirty minutes. The mg/mL reconstituted IgG4 Fc should be aliquoted to greater than 10 µg material per vial and stored at - 80°C. The inventors avoided repeated freeze-thaw cycles. [00509] IgG4 Fc was immobilized to target 550 RU on CM5 chip using amine coupling before this assay. hIgA1 was used as reference surface. [00510] The inventors equilibrated CM5 sensor chip and sodium acetate buffer to room temperature by leaving on bench for approximately twenty minutes.

131 30123-WO-PCT [00511] The inventors prepared 1x HBS-P running buffer by mixing 100 mL 10x HBS-P with 900 mL ultrapure water. Filter contents through a 0.22 µm PES membrane (Corning #431174). Fresh running buffer should be made up before every experiment. [00512] Surface immobilization: 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 amine coupling kit using volumes and rack orientation instructed by the prompts on the instrument protocol. Set target immobilization level to 550 RU. Surface plasmon resonance binding assay (single cycle kinetics) [00513] (1) With CM5 chip in the instrument, prime three times with 1x HBS-P running buffer. [00514] (2) Dilute test molecules to top concentration of 1 µM in 1x HBS-P. A minimum volume of 200 µL is recommended. Always use the same 1x HBS-P running buffer as the preparation primed through the system for dilution of test molecules. [00515] (3) In 96-well plate, dilute top concentration of test article 4-fold across six points. This dilution series can be achieved by aspirating 50 µL from the top concentration dose and mixing into 150 µL 1x HBS-P across and to the last (lowest concentration) point. [00516] (4) Add startup or blank conditions to appropriate wells as required by instrument protocol (1x HBS-P buffer only). [00517] (5) Prepare 50% dimethyl sulfoxide regeneration solution by mixing equal volumes of dimethyl sulfoxide and 1x HBS-P. [00518] (6) Allow protocol to run and fit affinity from single cycle kinetics trace. TABLE 22 Reagents and consumables Item Vendor Cat No Plastic vials 7 mm, 1000 pcs Cytiva BR100212 Rubber caps, type 3 (for 7 mm vials) Cytiva BR100502 Plastic vials, 15 mm Cytiva 29266981 Rubber caps, type 5 (for 15 mm vials) Cytiva BR100655 Microplate Foils, 96 Well Cytiva 28975816 10 mM acetate pH 5.0 Cytiva BR100351 Amine coupling kit Cytiva BR100050 Series S Sensor Chip CM5 Cytiva 29149603 PP Masterblock, 384 well (deep well plate) Greiner 781270

132 30123-WO-PCT TABLE 22 Reagents and consumables Item Vendor Cat No 1N sodium hydroxide Fisher Scientific SS277 HBS-P+ buffer, 10x Cytiva BR100671 Human IgG4 Fc protein, Tag Free (MALS & SPR verified) ACRO Biosystems IG4-H5205 Recombinant human IgA1 kappa BioRad HCA189 1L bottle top filter, 0.22 µm PES Corning 431174 96W Polypropylene v bottom plate Axygen P-96-450V-C [00519] Binding confirmation by surface plasmon resonance. The inventors performed surface plasmon resonance assays to confirm IgG4 Fc nanobody-binding. The inventors immobilized IgG4 Fc to a CM5 sensor chip by amine coupling right before the assays. The anti-IgG4 fragment was dosed 1 nM to 1 µM and characterized by single cycle kinetics with a contact time of sixty seconds, dissociation time of sixty seconds, and flow rate of 30 µL/minute. [00520] The inventors obtained IgG4, full length and fragment domains, labelled with, e.g., biotin, producing, e.g., a 12 kDa recombinant antibody fragment with biotin conjugation. The inventors also labelled IgG4 with AF647 (3.5-6 dye molecules/protein) for use in the surface plasmon resonance assays. [00521] The inventors tested unconjugated material in the surface plasmon resonance assays to make sure chemistry is not lowering the binding of the immunoglobulin region. TABLE 23 Components of the IgG4 assay Item Supplier Description CaptureSelect® Biotin anti- Thermo Fisher Catalog # 12 kDa recombinant antibody IgG4 (Human) Conjugate 7102902500 fragment w/biotin conjugation Human IgG4 Fc protein, Tag ACRO Biosystems Catalog # HEK293 expression AA Glu 99- Free IG4-H5205 Lys 327 Thermo nanobody, a commercial biotin-VHH fragment, is used as a positive control. [00522] The results of the surface plasmon resonance IgG4-degrader target-engagement assays confirmed binding. [00523] The inventors confirmed commercial anti-IgG4 nanobody binding to human IgG4 Fc. In n=2 characterization, K_D of about 20 nM was obtained. No binding response was observed for full-length IgG1-3 and IgG1-3 Fc regions at up to 1 µM concentrations.

133 30123-WO-PCT TABLE 24 Single cycle kinetics Run Steady State K_D (nM) First run, single cycle kinetics, steady state K_D (nM). 22 Second run, single cycle kinetics, steady state K_D (nM). 24 Nanobody (anti-IgG4) binding by surface plasmon resonance showed a K_D of 20nM. This result validates robust binding of the VHH nanobody to IgG4. [00524] The steady state affinity results for this surface plasmon resonance assay are consistent with the obtained single cycle kinetic runs. TABLE 25 Target engagement by surface plasmon resonance, single cycle kinetics. Degrader K_D (nM) K_D (nM) SPR K_D (nM) SPR K_D (nM) n=1 n=2 ABT310 (SEQ ID NO.31). VHH nanobody, 17 19 truncated hinge, Fc fusion, unconjugated. ABT311 (SEQ ID NO: 32). VHH nanobody, 13 ± 1 19 ± 1 10 10 unconjugated. ABT312 (SEQ ID NO: 33). VHH nanobody, 8.3 10 truncated hinge, unconjugated. AGT401. VHH w/o hinge region. βGN3 via 35 ± 10 20 ± 2 44 25 maleimide. AGT402. VHH-Fc fusion. βGN3 via MATE. 52 ± 13 64 39 AGT403. VHH with hinge region. βGN3 32 ± 3 34 29 via sortase. AGT404. VHH w/o hinge region. βGN3 via 19 ± 5 24 14 sortase. Degraders engage IgG4 with potent binding affinity surface plasmon resonance validates IgG4- degrader target-engagement. IgG4 degrader molecules are IgG subclass-selective, and do not engage IgG in any toxicology assay species. [00525] With this surface plasmon resonance method, the inventors demonstrated anti-IgG4 Fc nanobody ABT301 binding towards IgG4 Fc. Anti-IgG4 Fc nanobody ABT301 presents mid-nM binding affinity for IgG4 Fc.

134 30123-WO-PCT Additional assay information [00526] Preparation. The inventors prepared running buffer - 1X HBS-P was made by diluting 100 mL 10x HBS-P in 900 mL Milli-Q water. Buffer was filtered through a Corning 4311740.22 µm PES membrane. [00527] Sample preparation. Prepared 1 µM conjugate samples in HBS-P (same freshly made buffer to be used as running buffer in assay: [00528] Prepared 1000 µL of samples at a concentration of 1 µM in 1X HBS-P buffer using attached protocol calculations (also annotated above). Made up 200 µL for AGN402 to conserve material. [00529] (1) Added 150 µL 1X HBS-P to wells A1:F5 using multichannel pipette. [00530] (2) Added 200 µL 1 µM samples to appropriate wells. [00531] (3) Using multichannel pipette, transferred 50 µL from top dilution series and mixed in the following row to create a 1:4 serial dilution with 6 points from right to left. [00532] (4) Added 200 µL 1X HBS-P to well G1 as buffer control. [00533] (5) Mixed 1 mL DMSO with 1 mL 1X HBS-P to use as 50% DMSO regeneration buffer. Regen buffer was added to a 4 mL plastic vial and placed at position A1 of reagent rack 2, per above configuration. [00534] In Biacore S200 control software: Before screen, running buffer (1X HBS-P) primed three times by selecting "multi prime" in the tool menu. These selections were continued through template/wizard prompts to set up the protocol in Biacore S200 software. TABLE 26 Materials Vendor Catalogue # Echo 650 Beckman Coulter Biacore S200 GE Healthcare Rubber caps, type 3 (for 7 mm vials) Cytiva BR100502 Plastic vials, 15 mm Cytiva 29266981 Rubber caps, type 5 (for 15 mm vials) Cytiva BR100655 Microplate Foils, 96 Well Cytiva 28975816 Microplate Foils, 384 well Cytiva BR100577 HBS-N buffer, 10X Cytiva

135 30123-WO-PCT TABLE 26 Materials Vendor Catalogue # 10 mM acetate pH 4.5 Cytiva BR100350 10 mM acetate pH 5.0 Cytiva BR100351 10 mM acetate pH 5.5 Cytiva BR100352 Amine coupling kit Cytiva BR100050 Series S Sensor Chip CM5 Cytiva 29149603 PP Masterblock, 384 well (deep well plate) Greiner 781270 1N sodium hydroxide Fisher Scientific SS277 HBS-P+ buffer, 10x Cytiva BR100671 Human IgG4 Fc protein, Tag Free (MALS & SPR ACRO Biosystems IG4-H5205 verified) Normal human IgG control R&D Systems 1-001-A 1L bottle top filter, 0.22 µm PES Corning 431174 384 well PP 2.0 Microplate, Echo qualified Labcyte PP-0200 96W Polypropylene v bottom plate Axygen P-96-450V-C EXAMPLE X Subtype selectivity [00535] Anti-IgG4 Fc nanobody ABT301 does not show response to full-length IgG1, full-length IgG2, full-length IgG3, IgG1 Fc, IgG2 Fc, or IgG3 Fc at up to 1 µM concentrations. TABLE 27 Human isotype selectivity comparisons, as measured by surface plasmon resonance K_D, µM Compound Surface plasmon resonance KD (nM) Native Native Native Native hIgG hIgG2 hIgG3 hIgG4 hIgG4 hIgG1 hIgG2 hIgG3 hIgG4 1 Fc Fc Fc Fc Fc AGT401 >1 µM >1 µM >1 µM 36 >1 >1 >1 35 ± 0.025 µM µM µM 10 AGT402 >1 µM >1 µM >1 µM 15 >1 >1 >1 52 ± 0.001 µM µM µM 13 7 AGT403 >1 µM >1 µM >1 µM 33 >1 >1 >1 32 ± 0.027 µM µM µM 3

136 30123-WO-PCT TABLE 27 Human isotype selectivity comparisons, as measured by surface plasmon resonance K_D, µM Compound Surface plasmon resonance KD (nM) AGT404 >1 µM >1 µM >1 µM 43 >1 >1 >1 19 ± 0.052 µM µM µM 5 For native (FL) and Fc regions of IgG1, IgG2, and IgG3 subclasses, no binding response witnessed at 1 µM. KDs ± std. dev. reported from two independent surface plasmon resonance experiments, unless otherwise indicated. TABLE 28 Nonhuman isotype selectivity comparisons, as measured by surface plasmon resonance K_D, µM. IgG4 degraders do not demonstrate cross-species reactivity. Agent Recombinant Rhesus Cynomolgus Rhesus Rat IgG, Mouse IgG, Rabbit cynomolgus monkey monkey monkey whole whole IgG Fc monkey IgG1 IgG2 Fc IgG3 Fc IgG4 Fc molecule molecule AGT401 >1 µM >1 µM >1 µM 23 >1 µM >1 µM >1 µM AGT402 >1 µM >1 µM >1 µM 6.4 >1 µM >1 µM >1 µM AGT403 >1 µM >1 µM >1 µM 23 >1 µM >1 µM >1 µM AGT404 >1 µM >1 µM >1 µM 22 >1 µM >1 µM >1 µM Note: no binding response was observed for any IgG species (other than rhesus monkey IgG4 Fc) at 1 µM. Subsequent follow-up experiments for rhesus monkey IgG4 Fc were performed starting from 25 µM. [00536] The inventors demonstrated little cross species reactivity. The results demonstrated no engagement for non-human species, not even the rhesus monkey. Sequence alignment showed that IgG3 and IgG4 are similar in the binding site for the cynomolgus monkey sequence. EXAMPLE 21 Ternary complex assay to characterize on-mechanism pharmacological mode of activity. [00537] The inventors performed cell-based TR-FRET assays to assess ternary complex on cell surface. The objective of this assay was to screen IgG4 degrader conjugates in the FRET-based ternary complex formation assay previously developed using AlexaFluor647 labeled anti-hIgG4 as the FRET acceptor fluorophore. [00538] The TR-FRET principle is as follows: The donor fluorophore is excited with laser or flash lamp and emits light. The acceptor fluorophore excited by energy from donor if in proximity. The resulting signal emission from acceptor is proportional to level of interaction. See Li et. al., J. Biomol. Screen.,

137 30123-WO-PCT 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 evaluation to characterize pharmacological mechanism of action. The TR-FRET assay measures the induced proximity of the bifunctional degrader moieties to validate degrader-induced ASGPR-IgG4 proximity. [00539] The inventors used secondary antibody AF647 labeled anti-hIgG4 as the acceptor fluorophore in the TR-FRET assay. The condition for the assay were: 6.3 nM hIgG4 (Abcam), 25 nM ASGPR1, 2.5 nM streptavidin-europium cryptate, 20 nM AF-647 labeled anti-hIgG4, in 18 µL reaction, with 0.1% dimethyl sulfoxide. [00540] Assay: This assay was developed to confirm three-body ternary complex formation between ASGPR1 and IgG4 induced by a heterobifunctional degrader. An Echo acoustic liquid handler is used to dispense dimethyl sulfoxide negative control and AGN406 positive control, with handheld multichannel pipettes used to dispense the aqueous dilutions of test molecules. [00541] Assay preparation: The inventors slow thawed rASGPR from -80°C storage on ice. [00542] The inventors prepared AGN406 at a concentration of 12.5 μM in 100% dimethyl sulfoxide (100x final top concentration). The inventors added AGN406 and dimethyl sulfoxide to an Echo-qualified microplate for dispensing of positive and negative assay controls. [00543] The inventors diluted AGN406 two-fold, vertically (over sixteen points) in a 384-well v bottom plate by mixing 25 µL in 25 µL 1x buffer, from a top concentration of 250 μM in an Echo- qualified source plate. [00544] Assay 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, 0.01% TWEEN-20. TABLE 29 Example calculation for 40 mL of 1x buffer Component Stock Final Add HEPES pH 7.5 1000 mM 25 mM 1000 μL Sodium chloride 5000 mM 150 mM 1200 μL Calcium chloride 1000 mM 5 mM 200 μL Tween-20 10% 0.01 % 40 μL Dithiothreitol 1000 mM 1 mM 40 μL MilliQ water 37520 μL Total: 40000 μL

138 30123-WO-PCT [00545] (2) The inventors prepared a 50 μL degrader test article in 1x assay buffer at concentration of 30 μM as top dose in dilution series. [00546] (3) Using Echo liquid handler, the inventors dispensed 180 nL dimethyl sulfoxide (negative control), 180 nL 12.5 μM AGN406 (positive assay control), and 180 nL AGN406 dosed control to appropriate wells of a 384 well OptiPlate. Backfill these wells with 6 μL 1x assay buffer for volume scaling. [00547] (4) The inventors added 6 μL degrader dilutions to appropriate wells of assay plate using electronic multichannel repeating pipette. The plates were sealed and set aside. [00548] (5) The inventors prepared a 3x proteins mixture, which contains 75 nM ASGPR1 and 18.75 nM IgG4 in 1x assay buffer. TABLE 30 Example calculation Protein Stock Final 3x Add hIgG4 13.7 μM 0.00625 μM 0.01875 μM 6.84 μL rASGPR1-8 510 μM 0.025 μM 0.075 μM 0.74 μL 1x Buffer 4992.42 μL Total 5000.00 μL (6) Using Multidrop liquid handler, the inventors dispensed 6 μL proteins mixture to all wells of assay plate containing degrader dilutions and controls. [00550] (7) The inventors sealed the plate, centrifuged at 1000 RPM for one minute, and incubated for sixty minutes at room temperature. [00551] (8) The inventors prepared a 3x detection mix in 1x assay buffer: 7.5 nM streptavidin- Europium cryptate and 60 nM mouse anti-human IgG4 Fc-AlexaFluor647. TABLE 31 Example calculation for 5 mL solution Item Stock Final 3x Add Streptavidin Eu cryptate 733.3 nM 2.5 nM 7.5 nM 51.1 μL anti-hIgG4 AF647 3333 nM 20 nM 60 nM 90.0 μL 1x Assay Buffer 4858.9 μL Total 5000.0 μL [00552] (9) Using a Multidrop Combi liquid handler, the inventors dispensed 6 μL detection all wells of assay plate.

139 30123-WO-PCT [00553] (10) The inventors sealed the plate, centrifuged at 1000 RPM for one minute, and incubated for sixty minutes at room temperature. [00554] (11) The inventors recorded FRET using excitation at 320 nm and emission and 665 nm and 615 nm on an Envision multilabel reader. Data were outputted as a .csv file with FRET ratio data reduction already performed on reader. TR-FRET values were calculated as [(665/615)*1000] and plotted as the response at each test concentration of degrader. Assay signal to background was calculated as the mean signal of fixed concentration positive control (AGN406) divided by the mean signal of negative control (dimethyl sulfoxide) wells. Calculations for s/b and Z' were made. Dose- response data were formatted to plot 2 replicate values side by side. TABLE 32 Materials Item Vendor Catalog Number Lot (if applicable) Echo 650 Beckman Coulter E6XX-22020 Envision 2105 PerkinElmer 1050241 Mouse anti-human IgG4 Fc- Southern Biotech 9200-31 E1723-QB54B Alexa Fluor647 Streptavidin-Eu cryptate 20k Revvity 610SA KLB 37RA points TWEEN-20 Roche 73922900 11332465001 Calcium chloride 1 M solution Boston BioProducts MT-140 B01P105 Sodium chloride 5 M solution Boston BioProducts BM-244 K070112 DL-Dithiothreitol Sigma Aldrich D9779-5G SLCM3385 Dimethyl sulfoxide Sigma Aldrich D5879 Native Human IgG4 Protein Abcam AB183266 1075014-14 HEPES buffer, pH 7.51 M Boston BioProducts BBH-75 H100101 solution N-Avi-ASGPR1 (148-291) Viva Custom 2023.06.21 construct 8 (ASGPR1-8) 384 well PP 2.0 Microplate, Labcyte/Beckman PP-0200 Echo qualified ProxiPlate 384 PerkinElmer 6007290 [00555] Reagents for protein biotinylation for purification, immobilization or sensitive protein detection are commercially available for the AviTag™ Technology (GeneCopoeia, Rockville MD, USA). [00556] The inventors confirmed the In vitro activity of degrader-induced ASGPR-IgG4 proximity as measured by formation of a ternary complex by TR-FRET assays. Induced proximity between ASGPR and IgG4 generates a specific energy transfer signal. The TR-FRET signal is dependent on MoDE-induced proximity of ASGPR and IgG4. The TR-FRET signal of VHH-based degrader molecules validates the bifunctional mechanism of action. TABLE 33 In vitro characterization: TR-FRET validates degrader-induced IgG4-ASGPR proximity Binding moiety Ternary complex. TCF observed or not. ABT310 (SEQ ID NO.31). VHH nanobody, truncated hinge, Fc Not tested fusion, unconjugated. ABT311 (SEQ ID NO: 32). VHH nanobody, unconjugated. No (negative control) ABT312 (SEQ ID NO: 33). VHH nanobody, truncated hinge, Not tested unconjugated. AGT401. VHH w/o hinge region. βGN3 via maleimide. Yes AGT402. VHH-Fc fusion. βGN3 via MATE. Yes AGT403. VHH w/hinge region. βGN3 via sortase. Yes AGT404. VHH w/o hinge region. βGN3 via sortase. Yes [00557] The inventors titrated AGT406 across sixteen points in triplicate from 10 µM to 0.3 nM. [00558] The inventors used HEK293 fluorescent cell-based assays for assessing ternary complex formation. HEK293 fluorescent cell-based assays allow persons having ordinary skill in the biomedical art to monitor the kinetics of ternary complex formation in cells. [00559] The inventors also used other assays to assess event-driven mechanism of action. See, e.g., Pettersson & Crews, Drug Discovery Today Technologies, 31, 15-27 (Elsevier BV, 2019). This assay allows persons having ordinary skills in the biomedical art to monitor the kinetics of ternary complex formation in cells. [00560] The inventors also used NanoBRET, a bioluminescence resonance energy transfer (BRET) assay that measures protein-protein interactions (PPIs) in live cells. NanoBRET uses a small, bright luciferase called Nanoluc to tag intracellular proteins, and a long-wavelength fluorophore called HaloTag to act as an energy acceptor. When the two proteins are in close proximity, energy is transferred from

141 30123-WO-PCT Nanoluc to HaloTag, resulting in fluorescence emission at a specific wavelength. NanoBRET can be used to analyze binding events, signaling pathways, and receptor trafficking. EXAMPLE 23 Endocytosis assay for IgG4 compounds [00561] The inventors used IgG4 from Sydlabs (recombinant and already conjugated to fluorophore IF488). [00562] For routine culture of HEK-ASGPR1 cells, the inventors used DMEM high glucose, with 10% fetal bovine serum, 2 mM glutamine, and 1% P/S+200ug/ml geneticin. TABLE 34 Reagents for endocytosis assay Vendor Cat # (and Lot) DMEM high glucose with phenol Thermo Fisher (Gibco) 11960044 DMEM high glucose without phenol Thermo Fisher (Gibco) 31053028 Opti-MEM Reduced serum medium Thermo Fisher (Gibco) 31985062 Geneticin 50 mg/ml Thermo Fisher (Gibco) 10131-035 Fetal bovine serum, heat inactivated R&D systems (Optima) S12450H Glutamine Corning 25-005-CI Penicillin streptomycin Corning 30-002-CI Accutase Stemcell Technologies 07920 96-well black, flat clear bottom, poly-D Corning 3842 lysine coated Human IgG4 S228P isotype control- we Sydlabs PA007128 (conjugated to IF- asked them to conjugate this product to 488) IF-488

142 30123-WO-PCT TABLE 35 Agents tested mg/ml Molecular weight (Da) Calculated molarity (µM) AGN508 8.81 15636.9 563.41 AGN501 8.69 14498.8 599.36 AGN502 8.98 14570.8 616.30 AGN509 8.81 15321.6 575.01 AGN402 8.16 83779.5 97.40 [00563] The inventors used Dulbecco's phosphate-buffered saline without calcium/magnesium to rinse the cells once. [00564] Then, the inventors harvested HEK-ASGPR1 cells as follows: 5 ml accutase at T175 flasks 37°C for five minutes. Add 10 ml media and pipet up-down. Spin down 1000 rpm for five minutes. The inventors seeded 30k/well of a 96-well plate, 100 µl/well based on the plate map, using DMEM high glucose without phenol (31053028) +10% fetal bovine serum +2 mM glutamine +1% P/S+200 µg/ml geneticin. [00565] The inventors used poly-D lysine 96-well plates (Corning # 3842). [00566] The cells grew overnight in an incubator. The next day, the inventors ed 50nM IgG4 in the appropriate media (Opti-MEM+ 200 µg/ml G418 and compound).The inventors made an eight-point dose response with 500nM top concentration and two-fold dilution. For the last point, the dilution was ten-fold from the previous. [00567] The inventors incubated the assay overnight using an Incucyte instrument reading @ zero hours, four hours, six hours, ten hours, twelve hours, fourteen hours, sixteen hours, twenty hours, and twenty-four hours. [00568] At twenty-four hours, the inventors did a wash out with Dulbecco's phosphate-buffered saline with no calcium/magnesium to reduce the background. Then the inventors added 100 µl/well of media (Opti-MEM+ 200 µg/ml geneticin) and scanned gain at Incucyte. EXAMPLE 24 In vitro degradation assay. [00569] This on-mechanism degradation assay is by a direct, low-throughput Western blot measurement). Western assays can be used to measure the degradation in HEK293-ASGPR1 cells.

143 30123-WO-PCT Assay workflow [00570] Day 0. The inventors seeded cells at 0.5x10⁶/well 6-well plates. [00571] Day 1.100nM IgG4-AF488+60nM compound treatment overnight. [00572] Day 2. The inventors performed three washes with DPBS. The cells were lysed at T₀ hour (max) and other time points. The inventors added bafilomycin, which is a lysosome inhibitor, to some wells. [00573] Day 3. Lysed at the twenty-four hour point. TABLE 36 In vitro degradation assay results for AGT402 (Fc- fusion) Time point after % degradation (mean) wash - bafilomycin + bafilomycin One hour 55% 2% Two hours 79% 11% Five hours 94% 16% Twenty-four hours 98% 51% [00574] AGT402 can lead to almost complete IgG4 degradation by five hours post-wash. Bafilomycin inhibits degradation of IgG4 (degradation is lysosome-dependent). The ASGPR-dependent mechanism of Molecular Degraders of Extracellular targets (MoDE)-induced degradation is described by Caianiello, et al., Nature Chemical Biology (2021). Bafilomycin inhibits degradation of AF-488 conjugated IgG4 because the degradation is lysosome-dependent. EXAMPLE 25 LysoLgBiT assay [00575] The LysoLgBiT assay is a tool that can be used for studying the internalization and degradation of proteins of interest (POIs) in real time. By combining lysosome-specific signaling with protein labeling strategies, this assay delivers both precision and adaptability, making it suitable for a broad range of applications. A LgBiT Expression Vector and Stable Cell Line is commercially available from Promega (Catalog number N2681). See U.S. Pat. No.9,797,889 (Dixon et al.). [00576] Features of the LysoLgBiT assay include: [00577] LAMP1-LgBiT fusion protein, a lysosomal-specific fusion protein provides a robust and precise readout for the localization and degradation of HiBiT-tagged agents.

144 30123-WO-PCT [00578] The HiBiT tag, an 11-amino-acid peptide, can be appended to an agent using a simple and efficient copper-free click-chemistry method. This approach minimizes interference with the agent’s function and simplifies assay preparation. Fluorogenic protease substrates enable specificity by incorporating customizable linker sequences. [00579] The fusion of ASGPR1 HiBiT/NanoLuc proteins in HEK293 cells enables real-time monitoring of protein internalization kinetics. Luminescence reduction serves as a direct indicator of internalization and subsequent lysosomal degradation. [00580] Fluorophore/quencher pairs, such as DyLight 650 and IRDye QC-1, allow 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). [00581] The Incucyte assay provides a streamlined workflow with cost-effective protein labeling, optimized for high-throughput imaging. One pH sensitive dye, Incucyte FabFluor, is a Fab fragment conjugated to pH dependent fluorophore binds to Fc The dye is optimized for Incucyte optics and analysis software. [00582] Flow Cytometry offers high sensitivity with reduced background noise, achieved by gating on labeled cell populations. [00583] By appending the HiBiT tag, the LysoLgBiT assay can be applied to any agent. The HEK293 HiBiT/NanoLuc Fusion often needs no modification to a specific agent and has the potential for throughput scaling and broad utility, real time. EXAMPLE 26 In vitro toxicology studies [00584] Among the off-target assays that the inventors performed were measurements of agent cytotoxicity using an In vitro toxicology safety panel. The inventors performed the toxicology evaluation using HEPG2 cells, e.g., in a CellTiter-Glo assay. [00585] Also, metabolite identification (MetID) is a crucial and integral part of drug discovery and development. In this context, metabolism describes the breakdown and conversion of xenobiotics into molecules that can be excreted from the body. The inventors assessed MetID both in vitro and in vivo (blood, urine and liver) in mice. [00586] These results were sufficient to proceed with drug development. [00587] The inventors are also performing cell based screening assays to measure cytokine release.

145 30123-WO-PCT EXAMPLE 27 In vivo toxicology studies [00588] The inventors are performing toxicology assays to measure intravenous compatibility. [00589] Choice of toxicology species. The inventors are performing toxicology assays in several mammalian model species. [00590] Rat can be chosen as a second toxicology species. The inventors have experience with rat toxicology using testing by intravenous bolus and measuring cytokine reaction. [00591] Humanized IgG4 minipig can be chosen as a second toxicology species. The inventors can assess pharmacodynamics in the live minipig and collect minipig plasma to confirm binding to IgG4. [00592] A monkey species can be chosen as a second toxicology species because of similarity of immune system to humans. Rabbit only has one IgG isotype, so it is not as valuable. EXAMPLE 28 In vivo tolerability assays [00593] The inventors performed tolerability assays, starting with in vivo mouse assays (100 mg/kg), and then with in vivo rat assays (up to 1000 mg/kg). TABLE 37 Both Fc and non-Fc degraders well-tolerated in mouse at up to 100 mpk Agent Tolerability at 100 mpk (w/o target) AGT402 Well-tolerated AGT501 Well-tolerated AGT502 Well-tolerated TABLE 38 IgG4 + Agent (Fc-Fusion and Non-Fc-Fusion) Tolerability IgG4 (IV) Agent dose Agent route of Ratio # of Clinical signs Blood dose administration (Agent/ mice collection IgG4) 2 mg/kg Fc-fusion intravenous and 4:1 3 Mice appeared 1-2 (AGT402) 4.5 subcutaneous healthy 96 hours submandibular mg/kg of injection bleeds 2 mg/kg Fc-fusion intravenous 1:1 3 Mice appeared 1-2 (AGT402) healthy 96 hours submandibular 1.125 mg/kg of injection bleeds

146 30123-WO-PCT TABLE 38 IgG4 + Agent (Fc-Fusion and Non-Fc-Fusion) Tolerability IgG4 (IV) Agent dose Agent route of Ratio # of Clinical signs Blood dose administration (Agent/ mice collection IgG4) 2 mg/kg Non-fc-fusion intravenous and 4:1 3 Mice appeared 1-2 (AGT501, subcutaneous healthy 96 hours submandibular AGT502) 0.77 of injection bleeds mg/kg 2 or 4 - - - 3 Mice appeared 1-2 mg/kg healthy 96 hours submandibular of injection bleeds - Fc-fusion intravenous - 2 Mice appeared 1-2 (AGT402) up healthy submandibular to 100 mg/kg bleeds - Non-fc-fusion intravenous - 2 Mice appeared 1-2 (AGT501, healthy submandibular AGT502) up bleeds to 100 mg/kg 2 mg/kg Fc-fusion intravenous 1:1 3 Mice appeared 2 (AGT402) healthy 96 hours submandibular 1.125 mg/kg of injection bleeds 4 mg/kg Fc-fusion intravenous and 1:1 3 about 72 hours 2 (AGT402) 2.2 subcutaneous following submandibular mg/kg injections (2nd bleeds submandibular bleed), mice were in poor health, low motility, about 20% loss of body weight 4 mg/kg Fc-fusion intravenous 1:1 5 Mice appeared None (AGT402) 2.2 healthy and did mg/kg not lose weight about 100 hours after injection 4 mg/kg Fc-fusion intravenous 4:1 5 Mice appeared None (AGT402) 8.9 healthy and did mg/kg not lose weight about 100 hours after injection

147 30123-WO-PCT TABLE 38 IgG4 + Agent (Fc-Fusion and Non-Fc-Fusion) Tolerability IgG4 (IV) Agent dose Agent route of Ratio # of Clinical signs Blood dose administration (Agent/ mice collection IgG4) 4 mg/kg Non-fc-fusion intravenous 1:1 5 Mice appeared None (AGT501, healthy and did AGT502) 0.39 not lose weight mg/kg about 100 hours after injection 4 mg/kg Non-Fc-fusion intravenous 4:1 5 Mice appeared None (AGT501, healthy and did AGT502) 1.55 not lose weight mg/kg about 100 hours after injection 4 mg/kg Fc-fusion intravenous 1:1 3 Mice did not lose Three (AGT402) 2.2 weight 24 , 48, submandibular mg/kg 72, or 96 hours bleeds did not after injection affect motility or health of the mice Physical stability [00594] VHH-Fc fusion does not present issues in stability at 90 mg/mL in off-the-shelf biologics formulation (20 mM Histidine, pH 6.0, 8% sucrose). [00595] The inventors also confirmed in plasma stability assays. TABLE 39 Hold time study for protein intermediate Conditions Time points for pemphigus vulgaris batches Long-term -20±5℃ T₀, 0.5 M, 1 M Hold time tests for protein intermediate include Appearance, pH, Concentration, ELISA Binding, size exclusion chromatography, CE SDS (reducing and non-reducing), imaged capillary isoelectric focusing

148 30123-WO-PCT TABLE 40 Stability study for bioconjugate drug substance Stability study Conditions Time points for pemphigus vulgaris batches Long-term -20±5℃ T₀, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 36 months Accelerated 5±3℃, upright T₀, 1 month, 3 months, 6 months 5±3℃, inverted T₀, 1 month, 3 months, 6 months Stress 25±2℃ / 60±5% RH, T₀, 1 week, 2 weeks, 1 month, 3 months upright Stability tests for agent include appearance, pH, concentration, subvisible particles, ELISA, cell based potency, size exclusion chromatography, CE SDS (R/NR), imaged capillary isoelectric focusing, free drug levels, and excipient (e.g. PS-80) EXAMPLE 30 Other in vivo assays using mouse models. [00596] Pemphigus vulgaris model efficacy assay. Inject BVMAB786 mice subcutaneously. Day 1: Administer anti-Dsg3 IgG4 AutoAb, shortly followed by IgG4 degrader. Day 2: Administer ETA. Symptom onset after 18-72 hours. In presence of degrader, ETA should have no effect. In absence of degrader, ETA should induce pemphigus vulgaris symptoms. TABLE 41 Mouse models Origin, In vitro In vivo Reference subclass PVB28 Generated Dsg3 specific, Neonates dosed at 300 Di Zenzo, J. Clin. from Keratinocyte mg/mouse. + 2 mg ETA. Invest. (2012) (in untreated dissociation Symptom onset at eighteen vitro/vivo) patient, IgG4 assay hours. Qualitative analysis of cutaneous disease and histology MAB786 Generated Dsg3 specific, Neonates dosed at 0.6 Yeh, Clin. Imm. (AKA from Acantholysis mg/g. Symptom onset at (2006) F706) untreated induced in vitro eighteen hours. Cutaneous patient, IgG4 organ culture disease score and histology

149 30123-WO-PCT TABLE 42 Pemphigus vulgaris model efficacy assay. Body Antibody Subcutaneous Volume to Amount/ Dose solution weight (g) dose (mg/g) volume to inject inject/mouse (ml) mouse (mg) conc (mg/ml) (ml/kg) 25 0.6 10 0.25 15 60 [00597] Testing for antidrug antibodies (ADA) is done in mouse models. Many of the antibody drug antibodies are IgG4. [00598] Other assays using mice. In vivo biology to validate on-mechanism activity (pharmacokinetics/pharmacodynamics in ASGPR knock-out mouse). Pharmacokinetics and pharmacodynamics parameters are derived from intravenous and subcutaneous mouse studies. EXAMPLE 31 In vivo depletion assay [00599] In vivo characterization validated IgG4 depletion by VHH-ASGPR ligand bifunctional degraders (non-Fc fusion). Non-Fc fusion degraders also show IgG4-depletion. TABLE 43 Side-by-side comparison of pharmacodynamics profiles of Fc and non- Fc degraders versus unconjugated at twenty-four hours. Agent %-depletion of IgG4 AGT401 50% AGT402 60% ABT503 52% ABT504 63% In vivo characterization in mice validates IgG4-depletion. TABLE 44 % of depletion of antibodies in mice in vivo at eight hours and twenty-four hours, compared to IgG4 only Agent % Depletion (8 hours) % Depletion (24 hours) AGT401, conjugated 50.3% 52.6% AGT403, conjugated 52.2% 46.1% AGT404, conjugated 63.3% 58.4%

150 30123-WO-PCT TABLE 44 % of depletion of antibodies in mice in vivo at eight hours and twenty-four hours, compared to IgG4 only Agent % Depletion (8 hours) % Depletion (24 hours) ABT306 (SEQ ID NO: 11), unconjugated Negative-no difference 3.7% ABT304 (SEQ ID NO: 9), unconjugated Negative-no difference Negative-no difference ABT305 (SEQ ID NO: 10), unconjugated Negative-no difference Negative-no difference TABLE 45 Summary of Results: AGT402 (Fc-fusion) % Depletion relative to hIgG4 AUC Human Agent Agent Ratio AUC 8 AUC 24 AUC 48 AUC 72 AUC 96 IgG4 route of (mg/kg) (Agent/ hours hours hours hours hours (mg/kg) adminis- IgG4) tration PD1 2 mg/kg IV 4.5 4:1 87.3 60.4 N/A N/A N/A PD2 2 mg/kg IV 4.5 4:1 89.9 90.2 N/A 90.2 90.8 PD2 2 mg/kg subQ 4.5 4:1 4.2 27.3 N/A 59.0 65.4 PD3 2 mg/kg IV 4.5 4:1 90.6 91.8 92.6 PD3 2 mg/kg IV 1.125 1:1 88.9 85.5 86.3 PD4 4 mg/kg IV 2.2 1:1 89 89.4 88 87.5 87.8 PD4 4 mg/kg subQ 2.2 1:1 23.9 49.9 62 68.5 72 Intravenous administration of AGT402 depletes IgG4 levels by about 90% regardless of IgG4 dose or AGT402:IgG4 ratio. Subcutaneous administration of AGT402 depletes IgG4 levels by about 69%. Pharmacokinetics studies demonstrate about 50% subcutaneous bioavailability in mice. Pharmacodynamics and pharmacokinetics for non-Fc-fusion compounds. TABLE 46 In vivo characterization: degrader-dependent depletion of IgG4 (Fc fusion). Agent % depletion at 8 hours % depletion at 24 hours AGT402 87.3% 60.4% ABT310 Negative-no difference Negative-no difference Fc fusion degrader mediates IgG4-depletion at eight hours. Fc fusion degrader shows IgG4-depletion and rebound effect at twenty-four hours

151 30123-WO-PCT TABLE 47 %-depletion of IgG4 Agent Route of administration %-depletion IgG4 AGT402 subcutaneous 65% AGT402 intravenous 91% AGT501 intravenous 49% AGT502 intravenous 63% In vivo characterization validates IgG4-depletion by non-Fc-fusion (IV). In vivo characterization validates IgG4-depletion by Fc-fusion in mouse (intravenous and subcutaneous). In vivo characterization validates IgG4-depletion by non-Fc-fusion in mouse (intravenous) TABLE 48 Summary of Results: AGT501, AGT502 Agent Route of % IgG4 % IgG4 Pharmacokinetics Pharmacokinetics administration Depletion Depletion (Run 1) (Run 2) (Run1) (Run2) AGT501 intravenous 49% 61% Signal detected two Signal detected two hours post-dose hours post-dose AGT501 subcutaneous - 72% - Signal detected two hours post-dose AGT502 intravenous 63% 71% Signal detected two Signal detected two hours post-dose hours post-dose AGT502 subcutaneous - 87% - Signals detected two hours post-dose TABLE 49 %-depletion of IgG4 by VHH-Fc Route of administration Dose Frequency Dose (g) Depletion (24 hour) 2 mL subcutaneous Q1W 0.2 17% 2 mL subcutaneous BIW 0.2 50% (after 3 weeks) 6 mL subcutaneous Q1W 0.6 51% intravenous Q1W 0.4 68%

152 30123-WO-PCT EXAMPLE 32 In vivo bioavailability assays [00600] The inventors performed a set of assays in mouse and rat to provide insights into subcutaneous and intravenous administration routes with repeat dose regimens to show the bioavailability to a subject of the agents administered. [00601] Subcutaneous bioavailability: AGT402 Fc-fusion bifunctional degrader demonstrated approximately 50% bioavailability in a pharmacokinetics study in mice. [00602] Similarly, subcutaneous administration in rats has been used to measure the bioavailability of Fc and non-Fc agents, revealing potential correlations and distinctions in cross-species pharmacokinetics. [00603] Intravenous vs. subcutaneous administration: Comparative studies demonstrated the differences in bioavailability between intravenous and subcutaneous administration. [00604] The data for the four agents, including analysis of the rebound effect, demonstrate critical metrics for understanding dose-dependent behavior. [00605] Ongoing investigations are refining bioavailability data with additional time points collected beyond twenty-four and forty-eight hours to provide a comprehensive understanding of long-term systemic circulation. EXAMPLE 33 Analytical size exclusion chromatography assays [00606] The inventors used analytical size exclusion chromatography (SEC) assays to characterize and quantify the size distribution of agents within a drug substance sample. This assay detects agent aggregates and fragments. This information is used for quality control during development and manufacturing processes. All the analytical size exclusion chromatography assay results currently available look good. Developability data discovery indicates only a small decrease in size exclusion chromatography purity (<95%), shift towards acidic peaks by imaged capillary isoelectric focusing under thermal and pH-stressed conditions for VHH-Cys molecules. For VHH-Fc fusion molecules, the effects are less pronounced and limited to thermal stressed conditions. The data indicate Fc fusion confers stability advantage.

153 30123-WO-PCT EXAMPLE 34 Selecting human subjects for agent administration [00607] Outpatient studies can be performed for MuSK myasthenia gravis patients. Subcutaneous administration of the treatment, designed for at-home use, enhances patient convenience and compliance. The quantitative myasthenia gravis score offers a reliable metric to assess disease severity and therapeutic outcomes. The disease severity in MuSK myasthenia gravis correlates more closely with IgG4 autoantibody levels. MuSK myasthenia gravis can show a stronger clinical improvement response compared to classic myasthenia gravis. [00608] MuSK-specific IgG4 autoantibodies are a robust biomarker, providing a precise means of identifying eligible patients. This precision can assist with recruitment for clinical trials. TABLE 50 Summary Fc Fusion AGT402 VHH-Cys AGT501 VHH-Cys AGT502 Molecular weight (kDa) 83.8 14.5 14.6 Valency 2 1 1 Human stoichiometry dose 300-600 100 100 estimate (mg) Chemistry MATE conjugation Maleimide Maleimide thiosuccinimide thiosuccinimide Linker N/A G4S-Cys G3E-Cys-S Intravenous 91% 49% 63% pharmacodynamics: 96 hours % IgG4 depletion Subcutaneous 65 pharmacodynamics: 96 hours % IgG4 depletion Mouse tolerability w/o >100 >100 >100 target (mkg) pI 7.76 8.85 6.76 Surface plasmon resonance 20 19 20 K_D (nM) ASPGR Proximity Validated Validated Validated Endocytosis Validated Validated Validated In cell degradation Validated

154 30123-WO-PCT TABLE 50 Summary Fc Fusion AGT402 VHH-Cys AGT501 VHH-Cys AGT502 Passive transfer % depletion 69% (n=2) 72% (n=1) 87% (n = 1) (subcutaneous) Passive transfer % Depletion 90% (n=5) 55% (n=2) 67% (n = 2) (intravenous) Mouse bioavailability 50% Completed Completed (subcutaneous) Mouse tolerability (100 Well tolerated Well tolerated Well tolerated mg/kg) Integrated molecular (off Validation target protein binding). completed Validation completed for Fc fusion, Temperature/pH stability 90% size exclusion 90% size exclusion 90% size exclusion chromatography chromatography chromatography purity at 37°C purity at 37°C purity at 37°C iCIEF (freeze No change, only No change, change No change, change thaw/temperature and pH) changes at 37°C only in peaks under only in peaks under stressed conditions stressed conditions DLS-kD (aggregation) Indeterminate Low aggregation Low aggregation propensity propensity Concentratability (Targeted >75 mg/ml >75 mg/ml >75 mg/ml 75 mg/ml), in non-optimized formulation EXAMPLE 35 Human dose determination [00609] The inventors are developing a stoichiometric human dose determination. The inventors used the following premises for their first human dose determination: (1) IgG4 levels: 40 mg/dL; (2) IgG4 synthesis rate: 23 mg/day VHH: about 15 kDa; (3) VHH-Fc: about 80 kDa; (4) the maximum drug concentration is 100 mg/mL subcutaneous 50% BA; and (5) the maximum drug concentration is intravenous 100% BA. The inventors are refining human pharmacokinetics and pharmacodynamics parameters based on the rat pharmacokinetics/pharmacodynamics intravenous and subcutaneous assay results from the assays that are being performed. The inventors are also refining the human pharmacokinetics and pharmacodynamics parameters by comparing the human dose parameters with

155 30123-WO-PCT literature data. The inventors are confirming this methodology by. estimating ASGPR levels and kinetic parameters EXAMPLE 36 Agent effectiveness [00610] Persons having ordinary skills in the biomedical art can assess agent effectiveness using several criteria known to persons having ordinary skill in the biomedical art. Agent effectiveness can be determined by use of the Rey Auditory Verbal Learning Test (RAVLT). [00611] Agent effectiveness can be determined by use of the Montreal Overall Cognitive Assessment (MOCA). [00612] Agent effectiveness can be determined by the time to seizure freedom or cessation of status epilepticus without use of rescue therapy. EQUIVALENTS [00613] Persons having ordinary skill in the biomedical art will recognize or be able to determine using no more than routine experimentation many equivalents to the specific procedures described in this specification. 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 description and Examples in this specification can be employed. Furthermore, it is intended that specific items within lists of items, or subset groups of items within larger groups of items, can be combined with other specific items, subset groups of items or larger groups of items whether or not there is a specific disclosure in this specification identifying such a combination. A person of ordinary skill in the art will further recognize that the present inventive concept encompasses hydrated open- chain versions of the claimed compounds containing succinimide moieties that result from the ring- opening reactions of the succinimide moieties with water (i.e., hydrolysis). Such ring-opened structures and their alternative representations can be readily ascertained by a person of ordinary skill in the art. 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. [00614] Some embodiments of the invention can be practiced according to the following numbered paragraphs: [00615] 1. A composition of matter comprising: an IgG4-binding moiety, a cellular receptor-binding moiety capable of binding to hepatocytes or other degrading cells through asialoglycoprotein receptors

156 30123-WO-PCT (ASGPR) of hepatocytes or other cell receptors which are on surface degrading cells, and a linker moiety (optionally a single peptide linkage) connecting the IgG4-binding moiety and the cellular receptor- binding moiety. [00616] 2. The composition of matter of embodiment 1, wherein the IgG4-binding moiety is a published VHH affinity ligand variant antibody, a VHH affinity ligand variant, or an antigen-binding fragment thereof. [00617] 3. The composition of matter of embodiment 1, wherein the IgG4-binding moiety is a polypeptide having complementary determining regions (CDRs) of the anti-IgG4 VHH affinity ligand variant. [00618] 4. The composition of matter of embodiment 1, wherein the IgG4-binding moiety comprises three regions according to the Chothia numbering scheme having the structures of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

157 30123-WO-PCT [00619] 5. The composition of matter of embodiment 1, having a structure of: R^CN−(Xaa)y−R^CC, of , [AGN102] or a pharmaceutically acceptable salt thereof, wherein: each of a integer of 1 or greater; each AT is a IgG4-binding moiety or a fragment thereof; L is a linker moiety; and each TBT is independently a cellular receptor-binding moiety which binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a patient or subject, wherein the IgG4-binding moiety is a IgG4 VHH affinity ligand variant or an antigen-binding fragment thereof. [00621] 7. The composition of matter of embodiment 1, wherein the agent has the structure of a salt thereof, wherein the composition of

158 30123-WO-PCT [00622] 8. The composition of matter of embodiment 1, wherein the cellular receptor-binding moiety has the following or ; where R^A is a C₁-C₃ alkyl group optionally substituted with 1-5 halo, preferably fluoro, groups, preferably R^A is a methyl or ethyl group optionally substituted with from 1-3 fluoro groups; Z_A is -(CH₂)_IM, -O-(CH₂)_IM, S-(CH₂)_IM, NR_M-(CH₂)_IM, C(O)-(CH₂)_IM-, a PEG group containing from 1 to 8 preferably 1-4 ethylene glycol residues or a -C(O)(CH₂)_IMNR_M group (preferably a PEG containing group comprising from 1 to 8 ethylene glycol, preferably 2-4 ethylene glycol residues) where IM and R_M are the same as above; and Z_B is absent, (CH₂)_IM, C(O)-(CH₂)_IM- or C(O)- (CH₂)_IM-NR_M, where IM and R_M are the same as above. [00623] 9. A pharmaceutical composition comprising composition of matter of the preceding claims and a pharmaceutically acceptable excipient. [00624] 10. The composition of matter of embodiment 1, wherein the IgG4-binding moiety is a VHH affinity ligand variant or an antigen-binding fragment thereof. [00625] 11. The composition of matter of embodiment 1, wherein the agent has the structure of , [AGN102] or a pharmaceutically acceptable salt thereof, wherein: each of a integer of 1 or greater; each AT is a IgG4-binding moiety; L is a linker moiety; and each TBT is independently a cellular receptor-binding moiety which binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a patient or subject.

159 30123-WO-PCT [00626] 12. The composition of matter of embodiment 12, wherein the IgG4-binding moiety that binds to a specific amino acid residue of the antibody comprises [ABT101]. [00627] 13. A composition comprising: a first composition of matter comprising: an antibody moiety, a cellular receptor-binding moiety which binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a patient or subject, and a linker moiety linking the antibody moiety and the cellular receptor- binding moiety, and at least one additional composition of matter comprising a moiety capable of binding to the antibody that forms the antibody moiety of the first composition of matter. [00628] 14. The composition of embodiment 13, wherein the first compound has the structure of [AGN102] wherein AT, L, TBT, a and b are the same as those described in [00629] 15. The composition of embodiment 13, wherein the at least one additional agent has the structure of formula: LG−RG−L^RM(−TBT)_b, [AGN302]; LG−RG−H [MAT302]., or a combination thereof. [00630] 16. A pharmaceutical composition comprising the agent of any of embodiments 1-12. [00631] 17. A method of removing IgG4 in a patient or subject in need comprising administering to the subject or patient the agent of any of the embodiments 1-12. [00632] 18. A method of treating a disease state and/or condition associated with the upregulation of IgG4 in a patient or subject in need comprising administering to the subject or patient an effective amount of the agent of any of the embodiments 1-12. [00633] 19. A method of treating an IgG4-related disease (IgG4-RD) in a subject or patient in need comprising administering to the subject or patient an effective amount of the agent of any of the embodiments 1-12. REFERENCES [00634] Persons having ordinary skill in the biomedical art can use these patents, patent applications, and scientific references as guidance to predictable results when making and using the invention.

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162 30123-WO-PCT [00405] Deshpandeet al., Consensus statement on the pathology of IgG4-related disease. Modern Pathology.25(9), 1181–1192 (May 18, 2012). [00406] Di Zenzo et al., Pemphigus autoantibodies generated through somatic mutations target the desmoglein-3 cis-interface. The Journal of Clinical Investigation, 22(10), 3781-90 (October 1, 2012) (in vitro/vivo) [00407] Di Zenzo, Borradori, & Müller, The pathogenesis of pemphigus: Controversy versus complexity. Experimental Dermatology, 26(12), 1271-1273 (2017). [00408] Eming et al., Pathogenic IgG antibodies against desmoglein 3 in pemphigus vulgaris are regulated by HLA-DRB1*04:02-restricted T cells. J. Immunol., 193(9), 4391–9 (2022). Overall homology of Dsg3 between mice and humans are 85.6%. [00409] Emtenani, Hertl, Schmidt, & Hudemann, Mouse models of pemphigus: valuable tools to investigate pathomechanisms and novel therapeutic interventions. Frontiers in Immunology, 14, 1169947 (April 27, 2023). Lesions can be induced 18-72 hours following pemphigus vulgaris IgG administration. Continuous pemphigus vulgaris IgG administration promotes development of new erosions and crusts; however, skin lesions can heal if pemphigus vulgaris IgG injections are discontinued. [00410] Funakoshi et al., Enrichment of total serum IgG4 in patients with pemphigus. British Journal of Dermatology 167(6), 1245-1253 (2012). [00411] Guimaraes et al., Site-specific C-terminal and internal loop labeling of proteins using sortase- mediated reactions. Nature Protocols, 8, 1787 (September 2013). C-terminal sortase conjugation. [00412] Gupta et al., Nature Biomedical Engineering, 3, 917–929 (2019). [00413] Hudemann et al., IgG against the membrane-proximal portion of the desmoglein 3 ectodomain induces loss of keratinocyte adhesion, a hallmark in pemphigus vulgaris. Journal of Investigative Dermatology, 143(2),254-63 (February 1, 2023). Specificity to EC1 domain and Ca2+- regulated signaling are indicators of high pathogenicity. [00414] Hui et al., Crystal structure of deglycosylated human IgG4-Fc. Bioconjugate Chem., 26, 1456−1460 (2015). [00415] Huijbers et al. MuSK IgG4 autoantibodies cause myasthenia gravis by inhibiting binding between MuSK and Lrp4. Proc. Natl. Acad. Sci. U.S.A., 110(51), 20783-20788 (2013). [00416] Ishikawa et al., Cloning of the mouse desmoglein 3 gene (Dsg3): Interspecies conservation within the cadherin superfamily. Experimental Dermatology, 9(4), 229-39 (August 2000). Overall homology of Dsg3 between mice and humans are 85.6%. [00417] Kamisawa et al., IgG4-related disease. The Lancet.385 (9976), 1460–1471 (April 11, 2015).

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164 30123-WO-PCT [00431] Oh et al., Inhibition of sortase-mediated Staphylococcus aureus adhesion to fibronectin via fibronectin-binding protein by sortase inhibitors. Appl. Microbiol. Biotech., 70, 102-106 (March 2006). Sortase mediated conjugation. [00432] Pettersson & Crews, PROteolysis TArgeting Chimeras (PROTACs). Past present and future. Drug Discovery Today Technologies, Vol.31, pp.15-27 (Elsevier BV, 2019). [00433] Rodolico, Bonanno, Toscano, & Vita, MuSK-associated myasthenia gravis: Clinical features and management. Front. Neurol., 11, 660. Published 2020 Jul 23. doi:10.3389/fneur.2020.00660 [00434] Satou et al., Clinicopathological differential diagnosis of IgG4-related disease: A historical overview and a proposal of the criteria for excluding mimickers of IgG4-related disease. Pathol Int., 70(7), 391–402 (July 2020). [00435] Saxena & Wu, Advances in therapeutic Fc engineering–modulation of IgG-associated effector functions and serum half-life. Frontiers in immunology, 7, 580 (2016) (review). [00436] Schulze et al., An adult passive transfer mouse model to study desmoglein 3 signaling in pemphigus vulgaris. Journal of Investigative Dermatology 132(2), 346-55 (September 2011). AK23, a commonly IgG1 antibody used for investigations of pemphigus vulgaris. [00437] Shields et al., High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR. Journal of Biological Chemistry 276(9), 6591-6604 (2001). [00438] Stone, Zen, & Deshpande, IgG4-Related Disease. The New England Journal of Medicine. 366(6), 539–51 (February 2012). [00439] Strohl, Optimization of Fc-mediated effector functions of monoclonal antibodies. Current Opinion in Biotechnology, 20(6), 685-691 (2009). [00440] Takahashi et al., Experimentally induced pemphigus vulgaris in neonatal BALB/c mice: a time-course study of clinical, immunologic, ultrastructural, and cytochemical changes. J. Invest. Dermatol., 84(1), 41–6 (1985). Following a single i.p. injection of human pemphigus vulgaris IgG (examined with an electron microscope). [00441] Tamm & Schmidt, IgG binding sites on human Fcγ receptors. International reviews of immunology 16(1-2), 57-85 (1997). [00442] Theile et al., Site-specific N-terminal labeling of proteins using sortase-mediated reactions. Nature Protocols, 8, 2013 (2013). N-terminal sortase conjugation. [00443] Tilman et al., Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein Engineering, Design and Selection, Volume 29, Issue 10,

165 30123-WO-PCT pages 457–466 (October 2016). This paper shows that even LALA itself abolishes C1q binding. P329A alone is tested, and abolishes C1q binding, and reduces FcgR binding. They do not test P329A/LALA but show that P329G/LALA further reduces FcgR binding beyond LALA alone. [00444] Tucker et al., Isolation of state-dependent monoclonal antibodies against the 12- transmembrane domain glucose transporter 4 using virus-like particles. Proc. Natl. Acad. Sci. USA.115, E4990- E4999 (2018). [00445] Wallace et al., Predictors of disease relapse in IgG4-related disease following rituximab. Rheumatology, 55(6), 1000–1008 (June 6, 2016). [00446] Walsh, Experts Weigh in on IgG4 Disease - Diagnosis requires biopsy, and treatment starts with steroids. MedPage Today (April 1, 2015). [00447] Wen et al., Development of a FRET-based assay for analysis of mAbs internalization and processing by dendritic cells in preclinical immunogenicity risk assessment. AAPS J., 22(3), 68 (April 16, 2020). [00448] Yamada et al., AJICAP: Affinity peptide mediated regiodivergent functionalization of native antibodies. Angew Chem. Int. Ed Engl., 58(17), 5592-5597 (April 16, 2019). [00449] Yeh et al. Pathogenic human monoclonal antibody against desmoglein 3. Clinical Immunology.120(1), 68-75(2006). [00450] Yeh et al., Pathogenic human monoclonal antibody against desmoglein 3. Clin. Imm., 120(1), 68-75 (July 2006). PVMAB786 reacts with keratinocyte cell surfaces (similar to AK23). Intensity of binding increases between 3-6 hours and remains positive at twenty-four hours. [00451] Zalevsky et al. Enhanced antibody half-life improves in vivo activity. Nature Biotechnology, 28(2),157-159 (2010). Textbooks and technical references [00452] Current Protocols in Immunology (CPI) (2003). Coligan et al. (eds.) John Wiley and Sons, Inc. (ISBN 0471142735, 9780471142737). [00453] Current Protocols in Molecular Biology (CPMB), (2014). Ausubel (ed.), John Wiley and Sons (ISBN 047150338X, 9780471503385). [00454] Current Protocols in Protein Science (CPPS), (2005). Coligan (ed.), John Wiley and Sons, Inc. [00455] Immunology (2006). Werner Luttmann, published by Elsevier. [00456] Janeway's Immunobiology, (2014). Murphy, Mowat, & Weaver (eds.), Taylor & Francis Limited, (ISBN 0815345305, 9780815345305). [00457] Laboratory Methods in Enzymology: DNA, (2013). Lorsch (ed.) Elsevier (ISBN 0124199542).

166 30123-WO-PCT [00458] Lewin's Genes XI, (2014). published by Jones & Bartlett Publishers (ISBN-1449659055). [00459] Molecular Biology and Biotechnology: A Comprehensive Desk Reference, (1995). Meyers (ed.), published by VCH Publishers, Inc. (ISBN 1-56081-569-8). [00460] Molecular Cloning: A Laboratory Manual, 4th ed., Michael Richard Green and Joseph Sambrook, (2012). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (ISBN 1936113414). [00461] The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Porter et al., (eds.), published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908). [00462] The Merck Manual of Diagnosis and Therapy, 19^th edition (Merck Sharp & Dohme Corp., 2018). [00463] Remington’s, Pharmaceutical Sciences 23^rd edition (Elsevier, 2020). [00464] Smith & March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5^th edition (John Wiley & Sons, 2001). [00465] Larock, Comprehensive Organic Transformations, 2^nd edition (John Wiley & Sons, 1999). [00466] Greene & Wuts, Protecting Groups in Organic Synthesis, 3^rd edition (John Wiley & Sons, 1999). [00467] Throughout this application, several publications are referenced by author name and date, or by patent number or patent publication number. The disclosures of these publications are hereby incorporated in their entireties by reference into this application to describe the state of the art more fully as known to persons having ordinary skill in the biomedical art therein as of the date of the invention described and claimed in this specification. However, the citation of a reference in this specification should not be construed as an acknowledgement that this reference is prior art to the present invention. [00468] All patents and publications cited throughout this specification are incorporated by reference to disclose and describe the materials and methods that might be used with the technologies described in this specification. The publications discussed are provided only for their disclosure before the filing date. They should not be construed as an admission that the inventors may not antedate this disclosure under past invention or for any other reason. If there is an apparent discrepancy between a past patent or publication and the description given in this specification, the specification (including any definitions) and claims shall control. All statements about the date or contents of these documents are based on the information available to the applicants. These statements are no admission to the correctness of the dates or contents of these documents. The publication dates provided in this

167 30123-WO-PCT specification may differ from the actual publication dates. If there is an apparent discrepancy between a publication date in this specification and the actual publication date supplied by the publisher, the actual publication date shall control.

168 30123-WO-PCT SEQUENCE LISTING Sequence Number (ID): 1 Length: 122 Molecule Type: AA Features Location/Qualifiers: - source, 1..122 > mol_type, protein > note, ABT301. VHH nanobody > organism, synthetic construct - REGION, 26..32 > note, CDR-H1 Chothia scheme - REGION, 52..56 > note, CDR-H2 Chothia scheme - REGION, 98..105 > note, CDR-H3 Chothia scheme - SITE, 121 > note, Cysteine for linkage - SITE, 118 > note, Cysteine for linkage - REGION, 113..116 > note, TVSS marks the end of the VH domain - REGION, 118..122 > note, Hinge region Residues: QVQLQDSGGG LVQAGGSLRL SCEASGSIFS ISAMGWYRQA PGKQRELVAV ITTDGSTNYA 60 DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT DVYYCGASPR ALKWYWGQGT QVTVSSACPG 120 CA 122 Sequence Number (ID): 2 Length: 7 Molecule Type: AA Features Location/Qualifiers: - source, 1..7 > mol_type, protein > organism, Homo sapiens - REGION, 1..7

169 30123-WO-PCT > note, CDR-H1 of ABT301, amino acids 26-32 Residues: GSIFSIS 7 Sequence Number (ID): 3 Length: 5 Molecule Type: AA Features Location/Qualifiers: - source, 1..5 > mol_type, protein > organism, Homo sapiens - REGION, 1..5 > note, CDR-H2 of ABT301 amino acids 52-56 Residues: TTDGS 5 Sequence Number (ID): 4 Length: 8 Molecule Type: AA Features Location/Qualifiers: - source, 1..8 > mol_type, protein > organism, Homo sapiens - REGION, 1..8 > note, CDR-H3 of ABT301 amino acids 98-105 Residues: SPRALKWY 8 Sequence Number (ID): 5 Length: 22 Molecule Type: AA Features Location/Qualifiers: - source, 1..22 > mol_type, protein > note, C-term sortase tag > organism, synthetic construct Residues:

170 30123-WO-PCT GGGGSGGGGS LPETGGHHHH HH 22 Sequence Number (ID): 6 Length: 128 Molecule Type: AA Features Location/Qualifiers: - source, 1..128 > mol_type, protein > note, ABT311. VHH without hinge, (G4S)1-his6-C-term Cys, rigid C-term linker for the unpaired Cys for conjugation (4x Cys) > organism, synthetic construct - REGION, 98..105 > note, Complementarity-determining region 3 (Chothia) - REGION, 52..56 > note, Complementarity-determining region 2 (Chothia) - REGION, 26..32 > note, Complementarity-determining region 1 (Chothia) - REGION, 117..121 > note, G4S region for linkage - REGION, 122..127 > note, His6 region for linkage - SITE, 128 > note, Terminal cysteine for linkage Residues: QVQLQDSGGG LVQAGGSLRL SCEASGSIFS ISAMGWYRQA PGKQRELVAV ITTDGSTNYA 60 DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT DVYYCGASPR ALKWYWGQGT QVTVSSGGGG 120 SHHHHHHC 128 Sequence Number (ID): 7 Length: 154 Molecule Type: AA Features Location/Qualifiers: - source, 1..154 > mol_type, protein > note, ABT312. VHH nanobody-G4S-his6 > organism, synthetic construct - SIGNAL, 1..21 > note, Mouse Ig Kappa signal

171 30123-WO-PCT - REGION, 47..53 > note, CDR-H1 Chothia scheme - REGION, 73..77 > note, CDR-H2 Chothia scheme - REGION, 119..126 > note, CDR-H3 Chothia scheme - REGION, 124..127 > note, G4S linker - REGION, 129..134 > note, His6 tag Residues: METDTLLLWV LLLWVPGSTG DQVQLQDSGG GLVQAGGSLR LSCEASGSIF SISAMGWYRQ 60 APGKQRELVA VITTDGSTNY ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TDVYYCGASP 120 RALKWYWGQG TQVTVSSACP GCAGGGGSHH HHHH 154 Sequence Number (ID): 8 Length: 148 Molecule Type: AA Features Location/Qualifiers: - source, 1..148 > mol_type, protein > organism, synthetic construct - SIGNAL, 1..21 > note, Mouse Ig Kappa signal - REGION, 47..53 > note, CDR-H1 Chothia scheme - REGION, 73..77 > note, CDR-H2 Chothia scheme - REGION, 119..126 > note, CDR-H3 Chothia scheme - DOMAIN, 118..122 > note, G4S linker - DOMAIN, 123..138 > note, His6 tag Residues: METDTLLLWV LLLWVPGSTG DQVQLQDSGG GLVQAGGSLR LSCEASGSIF SISAMGWYRQ 60 APGKQRELVA VITTDGSTNY ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TDVYYCGASP 120

172 30123-WO-PCT RALKWYWGQG TQVTVSSGGG GSHHHHHH 148 Sequence Number (ID): 9 Length: 160 Molecule Type: AA Features Location/Qualifiers: - source, 1..160 > mol_type, protein > note, ABT304. VHH nanobody, G4S linker, with C-term Sortase site, His6 tag > organism, synthetic construct - SIGNAL, 1..21 > note, Mouse Ig Kappa signal - REGION, 47..53 > note, CDR-H1 Chothia scheme - REGION, 73..77 > note, CDR-H2 Chothia scheme - REGION, 119..126 > note, CDR-H3 Chothia scheme - DOMAIN, 124..127 > note, G4S linker - DOMAIN, 134..139 > note, His6 tag - DOMAIN, 128..133 > note, C-term Sortase site Residues: METDTLLLWV LLLWVPGSTG DQVQLQDSGG GLVQAGGSLR LSCEASGSIF SISAMGWYRQ 60 APGKQRELVA VITTDGSTNY ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TDVYYCGASP 120 RALKWYWGQG TQVTVSSACP GCAGGGGSLP ETGGHHHHHH 160 Sequence Number (ID): 10 Length: 154 Molecule Type: AA Features Location/Qualifiers: - source, 1..154 > mol_type, protein > note, ABT305. VHH nanobody with truncation of hinge-G4S-his6-C-term sortase > organism, synthetic construct

173 30123-WO-PCT - SIGNAL, 1..21 > note, Mouse Ig Kappa signal - REGION, 47..53 > note, CDR-H1 Chothia scheme - REGION, 73..77 > note, CDR-H2 Chothia scheme - REGION, 119..126 > note, CDR-H3 Chothia scheme Residues: METDTLLLWV LLLWVPGSTG DQVQLQDSGG GLVQAGGSLR LSCEASGSIF SISAMGWYRQ 60 APGKQRELVA VITTDGSTNY ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TDVYYCGASP 120 RALKWYWGQG TQVTVSSGGG GSLPETGGHH HHHH 154 Sequence Number (ID): 11 Length: 163 Molecule Type: AA Features Location/Qualifiers: - source, 1..163 > mol_type, protein > note, ABT306. VHH nanobody with truncation of hinge region-G4S-his6-rigid linker-Cys > organism, synthetic construct - SIGNAL, 1..21 > note, Mouse Ig Kappa signal - REGION, 47..53 > note, CDR-H1 Chothia scheme - REGION, 73..77 > note, CDR-H2 Chothia scheme - REGION, 119..126 > note, CDR-H3 Chothia scheme - DOMAIN, 118..122 > note, G4S linker - DOMAIN, 123..128 > note, His6 tag - DOMAIN, 129..143 > note, rigid linker + Cys Residues: METDTLLLWV LLLWVPGSTG DQVQLQDSGG GLVQAGGSLR LSCEASGSIF SISAMGWYRQ 60

174 30123-WO-PCT APGKQRELVA VITTDGSTNY ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TDVYYCGASP 120 RALKWYWGQG TQVTVSSGGG GSHHHHHHSP STPPTPSPST PPC 163 Sequence Number (ID): 12 Length: 374 Molecule Type: AA Features Location/Qualifiers: - source, 1..374 > mol_type, protein > note, ABT310. VHH without hinge, (G3ECS)1-Cys > organism, Homo sapiens - SIGNAL, 1..21 > note, Mouse Ig Kappa signal - REGION, 47..53 > note, CDR-H1 Chothia scheme - REGION, 73..77 > note, CDR-H2 Chothia scheme - REGION, 119..126 > note, CDR-H3 Chothia scheme - DOMAIN, 118..122 > note, G4S linker - DOMAIN, 123..374 > note, hIgG1 hinge (C>A)-Fc-LALA/PA Residues: METDTLLLWV LLLWVPGSTG DQVQLQDSGG GLVQAGGSLR LSCEASGSIF SISAMGWYRQ 60 APGKQRELVA VITTDGSTNY ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TDVYYCGASP 120 RALKWYWGQG TQVTVSSGGG GSEPKSADKT HTCPPCPAPE AAGGPSVFLF PPKPKDTLMI 180 SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 240 LNGKEYKCKV SNKALAAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY 300 PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH 360 NHYTQKSLSL SPGK 374 Sequence Number (ID): 13 Length: 120 Molecule Type: AA Features Location/Qualifiers: - source, 1..120

175 30123-WO-PCT > mol_type, protein > organism, Mus musculus - REGION, 117..120 > note, TVSS marks the end of the VH domain Residues: DVQLVESGGG LVQPGGSRKL SCAASGFTFS SFGMHWVRQA PEKGLEWVAY ISSGSSTLHY 60 ADTVKGRFTI SRDNPKNTLF LQMTSLRSED TGMYYCARWG NYPYYAMDYW GQGTSVTVSS 120 Sequence Number (ID): 14 Length: 13 Molecule Type: AA Features Location/Qualifiers: - source, 1..13 > mol_type, protein > organism, Homo sapiens - BINDING, 1..13 > note, VHH epitope on IgG4-Fc. The binding site on IgG4 was determined to be in two parts: Amino acids 47-57. Residues: DVSEQEDPEV QFN 13 Sequence Number (ID): 15 Length: 8 Molecule Type: AA Features Location/Qualifiers: - source, 1..8 > mol_type, protein > organism, Homo sapiens - BINDING, 1..8 > note, VHH epitope on IgG4-Fc. The binding site on IgG4 was determined to be- in two parts: Amino acids 106-114. Residues: SNKGPSSI 8 Sequence Number (ID): 16 Length: 6 Molecule Type: AA Features Location/Qualifiers:

176 30123-WO-PCT - source, 1..6 > mol_type, protein > organism, Homo sapiens Residues: ISAMGW 6 Sequence Number (ID): 17 Length: 14 Molecule Type: AA Features Location/Qualifiers: - source, 1..14 > mol_type, protein > organism, Homo sapiens Residues: YRQAPGKQRE LVAV 14 Sequence Number (ID): 18 Length: 11 Molecule Type: AA Features Location/Qualifiers: - source, 1..11 > mol_type, protein > organism, Homo sapiens Residues: ISRDNAKNTV Y 11 Sequence Number (ID): 19 Length: 13 Molecule Type: AA Features Location/Qualifiers: - source, 1..13 > mol_type, protein > organism, Homo sapiens Residues: YCGASPRALK WYW 13 Sequence Number (ID): 20

177 30123-WO-PCT Length: 9 Molecule Type: AA Features Location/Qualifiers: - source, 1..9 > mol_type, protein > organism, Homo sapiens Residues: VTVSSACPG 9 Sequence Number (ID): 21 Length: 21 Molecule Type: AA Features Location/Qualifiers: - source, 1..21 > mol_type, protein > organism, Homo sapiens Residues: DVYYXGASPR ALKWYWGQGT Q 21 Sequence Number (ID): 22 Length: 14 Molecule Type: AA Features Location/Qualifiers: - source, 1..14 > mol_type, protein > organism, Homo sapiens - BINDING, 1..14 > note, VHH binding region 1: Both Regions 1 and 2 likely involved in VHH binding. Residues: SIFSISAMGW YRQA 14 Sequence Number (ID): 23 Length: 22 Molecule Type: AA Features Location/Qualifiers: - source, 1..22 > mol_type, protein

178 30123-WO-PCT > organism, Homo sapiens - BINDING, 1..22 > note, VHH binding region 2: Both Regions 1 and 2 likely involved in VHH binding. Residues: AMGWYRQAPG KQRELVAVIT TD 22 Sequence Number (ID): 24 Length: 28 Molecule Type: AA Features Location/Qualifiers: - source, 1..28 > mol_type, protein > organism, Homo sapiens - BINDING, 1..28 > note, Qualifier ValueVHH binding region 1+2 Residues: SIFSISAMGW YRQAPGKQRE LVAVITTD 28 Sequence Number (ID): 25 Length: 20 Molecule Type: AA Features Location/Qualifiers: - source, 1..20 > mol_type, protein > organism, Homo sapiens - BINDING, 1..20 > note, VHH binding region 4: Ac-TDVYYCGASPRALKWYWGQGT-NH2 Residues: DVYYCGASPR ALKWYWGQGT 20 Sequence Number (ID): 26 Length: 330 Molecule Type: AA Features Location/Qualifiers: - source, 1..330 > mol_type, protein > organism, Homo sapiens

179 30123-WO-PCT Residues: ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPBPVTVS WNSGALTSGV HTFPAVLQSS 60 GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 120 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 180 STYRVVSVLT VLHQDWINGK EYKCRVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 240 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300 QQGNVFSCSV MHEATHNHYT QKSLSLSPGK 330 Sequence Number (ID): 27 Length: 326 Molecule Type: AA Features Location/Qualifiers: - source, 1..326 > mol_type, protein > organism, Homo sapiens Residues: ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPBPVTVS WNSGALTSGV HTFPAVLQSS 60 GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KCCVECPPCP APPVAGPSVF 120 LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFNWYVDG VEVHNAKTKP REBQFNSTFR 180 VVSVLTVLHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG OPREPQVYTL PPSREEMTKN 240 QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN 300 VFSCSVMHEA THNHYTQKSL SLSPGK 326 Sequence Number (ID): 28 Length: 327 Molecule Type: AA Features Location/Qualifiers: - source, 1..327 > mol_type, protein > organism, Homo sapiens Residues: ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPBPVTVS WNSGALTSGV HTFPAVLQSS 60 GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KTGPPCPSCP APEFLGGPSV 120 FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY 180 RVVSVLTVLH QDWINGKEYK CKVSNKGLPS SIEKTISKAK GOPREPQVYT LPPSQEEMTK 240 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG 300 NVFSCSVMHE ATHNHYTQKS LSLSLGK 327

180 30123-WO-PCT Sequence Number (ID): 29 Length: 133 Molecule Type: AA Features Location/Qualifiers: - source, 1..133 > mol_type, protein > note, ABT505. VHH w/o hinge, (G4S)1-his6-(G4S)1-Cys > organism, synthetic construct - SITE, 133 > note, Terminal cysteine for linkage - REGION, 128..132 > note, G4S region for linkage - REGION, 123..127 > note, His6 region for linkage - REGION, 117..121 > note, G4S region for linkage - REGION, 26..32 > note, Complementarity-determining region 1 (Chothia) - REGION, 52..56 > note, Complementarity-determining region 2 (Chothia) - REGION, 98..105 > note, Complementarity-determining region 3 (Chothia) Residues: QVQLQDSGGG LVQAGGSLRL SCEASGSIFS ISAMGWYRQA PGKQRELVAV ITTDGSTNYA 60 DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT DVYYCGASPR ALKWYWGQGT QVTVSSGGGG 120 SHHHHHHGGG GSC 133 Sequence Number (ID): 30 Length: 122 Molecule Type: AA Features Location/Qualifiers: - source, 1..122 > mol_type, protein > organism, synthetic construct - SITE, 122 > note, Terminal cysteine for linkage

181 30123-WO-PCT - REGION, 117..121 > note, G4S region for linkage - REGION, 26..32 > note, Complementarity-determining region 1 (Chothia) - REGION, 52..56 > note, Complementarity-determining region 2 (Chothia) - REGION, 98..105 > note, Complementarity-determining region 3 (Chothia) Residues: QVQLQDSGGG LVQAGGSLRL SCEASGSIFS ISAMGWYRQA PGKQRELVAV ITTDGSTNYA 60 DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT DVYYCGASPR ALKWYWGQGT QVTVSSGGGG 120 SC 122 Sequence Number (ID): 31 Length: 122 Molecule Type: AA Features Location/Qualifiers: - source, 1..122 > mol_type, protein > organism, synthetic construct - REGION, 117..122 > note, G3ECS region for linkage - SITE, 121 > note, Cysteine for linkage - REGION, 26..32 > note, Complementarity-determining region 1 (Chothia) - REGION, 52..56 > note, Complementarity-determining region 2 (Chothia) - REGION, 98..105 > note, Complementarity-determining region 3 (Chothia) Residues: QVQLQDSGGG LVQAGGSLRL SCEASGSIFS ISAMGWYRQA PGKQRELVAV ITTDGSTNYA 60 DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT DVYYCGASPR ALKWYWGQGT QVTVSSGGGE 120 CS 122 Sequence Number (ID): 32 Length: 127

182 30123-WO-PCT Molecule Type: AA Features Location/Qualifiers: - source, 1..127 > mol_type, protein > note, ABT504. VHH nanobody w/o hinge region-G4S-his6-Cys > organism, synthetic construct Residues: QVQLQDSGGG LVQAGGSLRL SCEASGSIFS ISAMGWYRQA PGKQRELVAV ITTDGSTNYA 60 DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT DVYYCGASPR ALKWYWGQGT QVTVSSGGGG 120 SHHHHHH 127 Sequence Number (ID): 33 Length: 324 Molecule Type: AA Features Location/Qualifiers: - source, 1..324 > mol_type, protein > note, Cynomolgus monkey IgG1 > organism, Macaca fascicularis Residues: SSASTKGPSV FPLAPSSRST SESTAALGCL VKDYFPEPVT VSWNSGSLTS GVHTFPAVLQ 60 SSGLYSLSSV VTVPSSSLGT QTYVCNVNHK PSNTKVDKRV EIKTCGGGSK PPTCPPCPAP 120 ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPD VKFNWYVNGA EVHHAQTKPR 180 ETQYNSTYRV VSVLTVTHQD WLNGKEYTCK VSNKALPAPI QKTISKDKGO PREPQVYTLP 240 PSREELTKNQ VSLTCLVKGF YPSDIVVEWE SSGOPENTYK TTPPVLDSDG SYFLYSKLTV 300 DKSRWGNVFS CSVMHEALHN HYTQ 324 Sequence Number (ID): 34 Length: 319 Molecule Type: AA Features Location/Qualifiers: - source, 1..319 > mol_type, protein > note, Cynomolgus monkey IgG2 > organism, Macaca fascicularis Residues: SSASTKGPSV FPLASCSRST SQSTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ 60

183 30123-WO-PCT SSGLYSLSSV VTVPSSSLGT QTYVCNVVHE PSNTKVDKTV GLPCRSTCPP CPAELLGGPS 120 VFLFPPKPKD TLMISRTPEV TCVVVDVSQE EPDVKFNWYV DGVEVHNAQT KPREEQFNST 180 YRVVSVLTVT HQDWLNGKEY TCKVSNKALP APRQKTVSKT KGQPREPQVY TLPPPREELT 240 KNQVSLTCLI KGFYPSDIVV EWASNGQPEN TYKTTPPVLD SDGSYFLYSK LTVDKSRWQQ 300 GNTFSCSVMH EALHNHYTQ 319 Sequence Number (ID): 35 Length: 325 Molecule Type: AA Features Location/Qualifiers: - source, 1..325 > mol_type, protein > note, Cynomolgus monkey IgG3 > organism, Macaca fascicularis Residues: SSASTKGPSV FPLVSCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ 60 SSGLYSLSSV VTVPSSSLGT QTYVCNVVHE PSNTKVDKRV EFTRPCDDTT PPCPPCPAPE 120 LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAQTKPRE 180 RQFNSTYRVV SVLTVTHQDW LNGKEYTCKV SNKGLPAPIE KTISKAKGQP REPQVYILPP 240 PQEELTKNQV SLTCLVTGFY PSDIAVEWES NGQPENTYKT TPPVLDSDGS YFLYSKLTVD 300 KSRWQQGNVF SCSVMHEALH NHYTQ 325 Sequence Number (ID): 36 Length: 317 Molecule Type: AA Features Location/Qualifiers: - source, 1..317 > mol_type, protein > note, Cynomolgus monkey IgG4 > organism, Macaca fascicularis Residues: SSASTKGPSV FPLASSSRST SESTVALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ 60 SSGLYSLSGV VTVPSSSLGT QTYVCNVVHQ PSNTKVDKRV EFTPPCPPCP APEVLGGPSV 120 FLFPPKPKDT LMISRIPEVT CVVVDVSEDP EVQFNWYVDG VEVHNAQTKP RERQFNSTYR 180 VVSVLTVTHQ DWLNGKEYTC KVSNKGLPAP IEKTISKAKG QPREPQVYIL PPPQEELTKN 240 QVSLTCLVTG FYPSDIAVEW ESNGQPENTY KTTPPMLDSD GSYLLYSKLT VNKSRWQPGN 300 IFTCSVMHEA LHNHYTQ 317

184 30123-WO-PCT Sequence Number (ID): 37 Length: 227 Molecule Type: AA Features Location/Qualifiers: - source, 1..227 > mol_type, protein > note, Rhesus monkey IgG1 > organism, Macaca mulatta Residues: TKVDKRVEIK TCGGGSKPPT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD 60 VSQEDPDVKF NWYVNGAEVH HAQTKPIYNS TYRVVSVLTV THQDWLNGKE YTCKVSNKAL 120 PAPIQKTISK DKGQPREPQV YTLPPSREEL TKNQVSLTCL VKGFYPSDIV VEWEQPENTY 180 KTTPPVLDSD GSYFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQ 227 Sequence Number (ID): 38 Length: 219 Molecule Type: AA Features Location/Qualifiers: - source, 1..219 > mol_type, protein > note, Rhesus monkey IgG2 > organism, Macaca mulatta Residues: TKVDKTVGLP CRSTCPPCPA ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEEPD 60 VKFNWYVDGV EVHNAQTKPF NSTYRVVSVL TVTHQDWLNG KEYTCKVSNK ALPAPRQKTV 120 SKTKGQPREP QVYTLPPSRE ELTKNQVSLT CLVKGFYPSD IVVEWAQPEN TYKTTPPVLD 180 SDGSYFLYSK LTVDKSRWQQ GNTFSCSVMH EALHNHYTQ 219 Sequence Number (ID): 39 Length: 225 Molecule Type: AA Features Location/Qualifiers: - source, 1..225 > mol_type, protein > note, Rhesus monkey IgG3 > organism, Macaca mulatta

185 30123-WO-PCT Residues: TKVDKRVEFT PPCGDTTPPC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV 60 SQEDPEVQFN WYVDGAEVHH AQTKPFNSTY RVVSVLTVTH QDWLNGKEYT CKVSNKGLPA 120 PIEKTISKAK GQPREPQVYI LPPPQEELTK NQVSLTCLVT GFYPSDIAVE WEQPENTYKT 180 TPPVLDSDGS YFLYSKLTVD KSRWQQGNTF SCSVMHEALH NHYTQ 225 Sequence Number (ID): 40 Length: 218 Molecule Type: AA Features Location/Qualifiers: - source, 1..218 > mol_type, protein > note, Rhesus monkey IgG1 > organism, Macaca mulatta Residues: TKVDKRVEFT PPCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV 60 QFNWYVDGAE VHHAQTKPFN STYRVVSVLT VTHQDWLNGK EYTCKVSNKG LPAPIEKTIS 120 KAKGQPREPQ VYILPPPQEE LTKNQVSLTC LVTGFYPSDI AVEWEQPENT YKTTPPVLDS 180 DGSYFLYSKL TVDKSRWQPG NIFTCSVMHE ALHNHYTQ 218 Sequence Number (ID): 41 Length: 314 Molecule Type: AA Features Location/Qualifiers: - source, 1..314 > mol_type, protein > organism, Mus musculus Residues: LAPGSAAQTN SMVTLGCLVK GYFPEPVTVT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV 60 PSSTWPSQTV TCNVAHPASS TKVDKKIVPR DCGCKPCICT VPEVSSVFIF PPKPKDVLTI 120 TLTPKVTCVV VDISKDDPEV QFSWFVDDVE VHTAQTKPRE EQINSTFRSV SELPIMHQDW 180 LNGKEFKCRV NSAAFPAPTE KTISKTKGRP KAPQVYTIPP PKEQMAKDKV SLTCMITNFF 240 PEDITVEWQW NGQPAENYKN TQPIMDTDGS YFVYSKLNVQ KSNWEAGNTF TCSVLHEGLH 300 NHHTEKSLSH SPGK 314 Sequence Number (ID): 42 Length: 320

186 30123-WO-PCT Molecule Type: AA Features Location/Qualifiers: - source, 1..320 > mol_type, protein > organism, Mus musculus Residues: LAPVCGDTTG SSVTLGCLVK GYFPEPVTLT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV 60 TSSTWPSQSI TCNVAHPASS TKVDKKIEPR GPTIKPCPPC KCPAPNLLGG PSVFIFPPKI 120 KDVLMISLSP IVTCVVVDVS EDDPDVQISW FVNNVEVHTA QTQTHREDYN STLRVVSALP 180 IQHQDWMSGK EFKCKVNNKD LPAPIERTIS KPKGSVRAPQ VYVLPPPEEE MTKKQVTLTC 240 MVTDFMPEDI YVEWTNNGKT ELNYKNTEPV LDSDGSYFMY SKLRVEKKNW VERNSYSCSV 300 VHEGLHNHHT TKSFSRTPGK 320 Sequence Number (ID): 43 Length: 259 Molecule Type: AA Features Location/Qualifiers: - source, 1..259 > mol_type, protein > organism, Mus musculus Residues: LAPGCGDTTG SSVTLGCLVK GYFPESVTVT WNSGSLSSSV HTFPALLQSG LYTMSSSVTV 60 PSSTWPSQTV TCSVAHPASS TTVDKKLEPS GPISTINPCP PCKECHKCPA PNLEGGPSVF 120 IFPPNIKDVL MISLTPKVTC VVVDVSEDDP DVQISWFVNN VEVHTAQTQT HREDYNSTIR 180 VVSTLPIQHQ DWMSGKEFKC KVNNKDLPSP IERTISKIKG LVRAPQVYIL PPPAEQLSRK 240 DVSLTCLVVG FNPGDISVE 259 Sequence Number (ID): 44 Length: 253 Molecule Type: AA Features Location/Qualifiers: - source, 1..253 > mol_type, protein > organism, Mus musculus Residues: LVPGCGDTSG SSVTLGCLVK GYFPEPVTVK WNYGALSSGV RTVSSVLQSG FYSLSSLVTV 60 PSSTWPSQTV ICNVAHPASK TELIKRIEPR IPKPSTPPGS SCPPGNILGG PSVFIFPPKP 120

187 30123-WO-PCT KDALMISLTP KVTCVVVDVS EDDPDVHVSW FVDNKEVHTA WTOPREAQYN STFRVVSALP 180 IQHQDWMRGK EFKCKVNNKA LPAPIERTIS KPKGRAQTPQ VYTIPPPREO MSKKKVSLTC 240 LVTNFFSEAI SVE 253 Sequence Number (ID): 45 Length: 316 Molecule Type: AA Features Location/Qualifiers: - source, 1..316 > mol_type, protein > organism, Rattus norvegicus Residues: LAPGTALKSN SMVTLGCLVK GYFPEPVTVT WNSGALSSGV HTFPAVLQSG LYTLTSSVTV 60 PSSTWSSQAV TCNVAHPASS TKVDKKIVPR NCGGDCKPCI CTGSEVSSVF IFPPKPKDVL 120 TITLTPKVTC VVVDISQDDP EVHFSWFVDD VEVHTAQTRP PEEQFNSTFR SVSELPILHQ 180 DWLNGRTFRC KVTSAAFPSP IEKTISKPEG RTQVPHVYTM SPTKEEMIQN EVSITCMVKG 240 FYPPDIYVEW QMNGQPQENY KNTPPTMDTD GSYFLYSKLN VKKEKWQQGN TFTCSVLHEG 300 LHNHHTEKSL SHSPGK 316 Sequence Number (ID): 46 Length: 312 Molecule Type: AA Features Location/Qualifiers: - source, 1..312 > mol_type, protein > organism, Rattus norvegicus Residues: LAPGTALKSN SMVTLGCLVK GYFPEPVTVT WNSGALSSGV HTFPAVLQSG LYTLTSSVTV 60 PSSTWSSQAV TCNVAHPASS TKVDKKIVPR ECNPCGCTGS EVSSVFIFPP KTKDVLTITL 120 TPKVTCVVVD ISQNDPEVRF SWFIDDVEVH TAQTHAPEKQ SNSTLRSVSE LPIVHRDWLN 180 GKTFKCKVNS GAFPAPIEKS ISKPEGTPRG PQVYTMAPPK EEMTQSQVSI TCMVKGFYPP 240 DIYTEWKMNG QPQENYKNTP PTMDTDGSYF LYSKLNVKKE TWQQGNTFTC SVLHEGLHNH 300 HTEKSLSHSP GK 312 Sequence Number (ID): 47 Length: 326 Molecule Type: AA

188 30123-WO-PCT Features Location/Qualifiers: - source, 1..326 > mol_type, protein > organism, Rattus norvegicus Residues: LAPGCGDTTS STVTLGCLVK GYFPEPVTVT WNSGPLSSDV HTFPAVLQSG LYTLTSSVTV 60 PSSTWPSQTV TCNVAHPASS TKVDKKIERR NGGIEPKPIP TCPTCHKCPA PELLGGPSVF 120 IFPPKPKDIL LISQNAKVTC VVVDVSEEEP DVQFSWFVNN VEVHTAQTQP REEQYNSTFR 180 VVSALPIQHQ DWMSGKEFKC KVNNKALPSP IEKTISKPKG LVRKPQVYVM GPPTEQLTEK 240 AVILTCLITD FLPNNIGVEW TSNGRIEKNY KNTEPVMDSD GSFFMYSKLN VERSRWDSRA 300 SFVCSVVHEG LHNRHVEKSI SRPPGK 326 Sequence Number (ID): 48 Length: 319 Molecule Type: AA Features Location/Qualifiers: - source, 1..319 > mol_type, protein > organism, Rattus norvegicus Residues: LVPGCSGTSG SLVTLGCLVK GYFPEPVTVK WNYGALSSGV HTFPAVLQSG LYTLSSSVTV 60 PSSTWSSQTV TCSVAHPATK SDLIKRIEPR RPKPRPPTDI CSCDDNLGRP SVFIFPPKPK 120 DTLMITLTPK VTCVVVDVSE EEPDVQFSWF VDNIRVFTAQ TQPHEEQLNG TFRVVSTLHI 180 QHQDWMSGKE FKCKVNNKDL PSPIEKTISK PRGKARTPQV YTIPPPREQM SKNKVSLTCM 240 VTSFYPASIS VEWERNGELE ODYKNTLPVL DSDESYFLYS KLSVDTDSWM RGDIYTCSVV 300 HEALHNHHTQ KNLSRSPGK 319 Sequence Number (ID): 49 Length: 202 Molecule Type: AA Features Location/Qualifiers: - source, 1..202 > mol_type, protein > organism, Canis lupus Residues: DILRITRTPE VTCVVLDLGR EDPEVQISWF VDGKEVHTAK TQSREQQFNG TYRVVSVLPI 60 EHQDWLTGKE FKCRVNHIDL PSPIERTISK ARGRAHKPSV YVLPPSPKEL SSSDTVSITC 120

189 30123-WO-PCT LIKDFYPPDI DVEWQSNGQQ EPERKHRMTP PQLDEDGSYF LYSKLSVDKS RWQQGDPFTC 180 AVMHETLQNH YTDLSLSHSP GK 202 Sequence Number (ID): 50 Length: 201 Molecule Type: AA Features Location/Qualifiers: - source, 1..201 > mol_type, protein > organism, Canis lupus Residues: DTLLIARTPE VTCVVVDLDP EDPEVQISWF VDGKQMQTAK TQPREEQFNG TYRVVSVLPI 60 GHQDWLKGKQ FTCKVNNKAL PSPIERTISK ARGQAHQPSV YVLPPSREEL SKNTVSLTCL 120 IKDFFPPDID VEWQSNGQQE PESKYRTTPP OLDEDGSYFL YSKLSVDKSR WORGDTFICA 180 VMHEALHNHY TOESLSHSPG K 201 Sequence Number (ID): 51 Length: 201 Molecule Type: AA Features Location/Qualifiers: - source, 1..201 > mol_type, protein > organism, Canis lupus Residues: DILVTARTPT VTCVVVDLDP ENPEVQISWF VDSKQVQTAN TQPREEQSNG TYRVVSVLPI 60 GHQDWLSGKQ FKCKVNNKAL PSPIEEIISK TPGQAHQPNV YVLPPSRDEM SKNTVTLTCL 120 VKDFFPPEID VEWQSNGQQE PESKYRMTPP OLDEDGSYFL YSKLSVDKSR WORGDTFICA 180 VMHEALHNHY TOISLSHSPG K 201 Sequence Number (ID): 52 Length: 202 Molecule Type: AA Features Location/Qualifiers: - source, 1..202 > mol_type, protein > organism, Canis lupus Residues:

190 30123-WO-PCT DILRITRTPE ITCVVLDLGR EDPEVQISWE VDGKEVHTAK TOPREQQFNS TYRVVSVLPI 60 EHQDWLTGKE FKCRVNHIGL PSPIERTISK ARGQAHQPSV YVLPPSPKEL SSSDTVTLTC 120 LIKDFFPPEI DVEWQSNGQP EPESKYHTTA PQLDEDGSYF LYSKLSVDKS RWQQGDTFTC 180 AVMHEALQNH YTDLSLSHSP GK 202 Sequence Number (ID): 53 Length: 227 Molecule Type: AA Features Location/Qualifiers: - source, 1..227 > mol_type, protein > organism, Sus scrofa Residues: TKPPCPICPG CEVAGPSVFI FPPKPKDTLM ISQTPEVTCV VVDVSKEHAE VQFSWYVDGV 60 EVHTAETRPK EEQFNSTYRV VSVLPIQHQD WLKGKEFKCK VNNVDLPAPI TRTISKAIGQ 120 SREPQVYTLP PPAEELSRSK VTVTCLVIGF YPPDIHVEWK SNGQPEPEGN YRTTPPQQDV 180 DGTFFLYSKL AVDKARWDHG ETFECAVMHE ALHNHYTQKS ISKTQGK 227 Sequence Number (ID): 54 Length: 227 Molecule Type: AA Features Location/Qualifiers: - source, 1..227 > mol_type, protein > organism, Sus scrofa Residues: TKPPCPICPA CEGPGPSAFI FPPKPKDTLM ISRTPKVTCV VVDVSQENPE VQFSWYVDGV 60 EVHTAQTRPK EEQFNSTYRV VSVLPIQHQD WLNGKEFKCK VNNKDLPAPI TRIISKAKGQ 120 TREPQVYTLP PPTEELSRSK VTLTCLVTGF YPPDIDVEWQ RNGQPEPEGN YRTTPPQQDV 180 DGTYFLYSKL AVDKASWQRG DTFQCAVMHE ALHNHYTQKS IFKTPGK 227 Sequence Number (ID): 55 Length: 233 Molecule Type: AA Features Location/Qualifiers: - source, 1..233 > mol_type, protein

191 30123-WO-PCT > organism, Sus scrofa Residues: EPPTPICPEI CSCPAAEVLG APSVFLFPPK PKDILMISRT PKVTCVVVDV SQEEAEVQFS 60 WYVDGVQLYT AQTRPMEEQF NSTYRVVSVL PIQHQDWLKG KEFKCKVNNK DLLSPITRTI 120 SKATGPSRVP QVYTLPPAWE ELSKSKVSIT CLVTGFYPPD IDVEWQSNGQ QEPEGNYRTT 180 PPQQDVDGTY FLYSKLAVDK VRWQRGDLFQ CAVMHEALHN HYTQKSVSQT PGK 233 Sequence Number (ID): 56 Length: 227 Molecule Type: AA Features Location/Qualifiers: - source, 1..227 > mol_type, protein > organism, Sus scrofa Residues: TKPPCPICPA CEGPGPSAFI FPPKPKDTLM ISRTPKVTCV VVDVSQENPE VQFSWYVDGV 60 EVHTAQTRPK EEQFNSTYRV VSVLPIQHQD WLNGKEFKCK VNNKDLPAPI TRIISKAKGQ 120 TREPQVYTLP PPTEELSRSK VTLTCLVTGF YPPDIDVEWQ RNGQPEPEGN YRTTPPQQDV 180 DGTYFLYSKL AVDKASWQRG DTFQCAVMHE ALHNHYTQKS IFKTPGK 227 Sequence Number (ID): 57 Length: 223 Molecule Type: AA Features Location/Qualifiers: - source, 1..223 > mol_type, protein > organism, Sus scrofa Residues: PCPICPGCEV AGPSVFIFPP KPKDILMISR TPEVTCVVVD VSKEHAEVQF SWYVDGVEVH 60 TAETRPKEEQ FNSTYRVVSV LPIQHEDWLK GKEFECKVNN EDLPGPITRT ISKAKGVVRS 120 PEVYTLPPPA EELSKSKVTL TCLVKSFFPP FIHVGWKING QLEPEGNYHT TPPQEDEDGT 180 YFLYSKLAVD KARLQSGGIH CAVMHEALHN HYTEKSVSLT PGK 223 Sequence Number (ID): 58 Length: 224 Molecule Type: AA Features Location/Qualifiers:

192 30123-WO-PCT - source, 1..224 > mol_type, protein > organism, Sus scrofa Residues: PCPICPACEG PGPSAFIFPP KPKDTLMISR TPKVTCVVVD VSQENPEVQF SWYVDGVEVH 60 TAQTRPKEEQ FNSTYRVVSV LPIQHQDWLN GKEFKCKVNN KDLPAPITRI ISKAKGQTRE 120 PQVYTLPPPT EELSRSKLSV TCLITGFYPP DIDVEWQRNG QPEPEGNYRT TPPQQDVDGT 180 YFLYSKLAVD KASWQRGDPF QCAVMHEALH NHYTQKSIFK TPGN 224 Sequence Number (ID): 59 Length: 133 Molecule Type: AA Features Location/Qualifiers: - source, 1..133 > mol_type, protein > note, ABT501. VHH nanobody w/o hinge region-G4S-his6-G4S-Cys > organism, synthetic construct - SITE, 133 > note, Terminal cysteine for linkage - REGION, 128..132 > note, G4S region for linkage - REGION, 125..131 > note, His6 region for linkage - REGION, 115..120 > note, G4S region for linkage Residues: QVQLQDSGGG LVQAGGSLRL SCEASGSIFS ISAMGWYRQA PGKQRELVAV ITTDGSTNYA 60 DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT DVYYCGASPR ALKWYWGQGT QVTVSSGGGG 120 SHHHHHHGGG GSC 133 Sequence Number (ID): 60 Length: 154 Molecule Type: AA Features Location/Qualifiers: - source, 1..154 > mol_type, protein > note, ABT507. Full sequence for sortase linkage with shortened leader sequence.

193 30123-WO-PCT > organism, synthetic construct - REGION, 1..17 > note, shortened leader sequence - REGION, 149..154 > note, his6 for conjugation Residues: MGWSCILFLV ATATGHSQVQ LQDSGGGLVQ AGGSLRLSCE ASGSIFSISA MGWYRQAPGK 60 ORELVAVITT DGSTNYADSK GFTISRDNAK NTVYLQMNSL KPEDTDVYYC GASPRALKWY 120 WGQGTQVTVS SACPGCAGGG GSLPETGGHH HHHH 154 Sequence Number (ID): 61 Length: 151 Molecule Type: AA Features Location/Qualifiers: - source, 1..151 > mol_type, protein > note, ABT508. Truncated sequence for sortase linkage with shortened leader sequence > organism, synthetic construct - REGION, 1..19 > note, shortened leader sequence - REGION, 146..151 > note, his6 for conjugation Residues: MGWSCIILFL VATATGVHSQ VQLQDSGGGL VQAGGSLRLS CEASGSIFSI SAMGWYROAP 60 GKORELVAVI TTDGSTNYAD SKGRFTISRD NAKNTVYLQM NSLKPEDTDV YYCGASPRAL 120 KWYWGQGTQV TVSSGGGGSL PETGGHHHHH H 151 Sequence Number (ID): 62 Length: 161 Molecule Type: AA Features Location/Qualifiers: - source, 1..161 > mol_type, protein > note, ABT509. Truncated sequence for maleimide beta-GN3 linage with shortened leader sequence > organism, synthetic construct - REGION, 1..19

194 30123-WO-PCT > note, shortened leader sequence - REGION, 141..146 > note, his6 for conjugation - SITE, 161 > note, terminal Cys for conjugation Residues: MGWSCIILFL VATATGVHSQ VQLQDSGGGL VQAGGSLRLS CEASGSIFSI SAMGWYRQAP 60 GKQRELVAVI TTDGSTNYAD SVKGRFTISR DNAKNTVYLQ MNSLKPEDTD VYYCGASPRA 120 LKWYWGQGTQ VTVSSGGGGS HHHHHHSPST PPTPSPSTPP C 161 Sequence Number (ID): 63 Length: 122 Molecule Type: AA Features Location/Qualifiers: - source, 1..122 > mol_type, protein > note, ABT503. VHH nanobody with truncation of hinge region-G3E-Cys-S > organism, synthetic construct Residues: QVQLQDSGGG LVQAGGSLRL SCEASGSIFS ISAMGWYRQA PGKQRELVAV ITTDGSTNYA 60 DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT DVYYCGASPR ALKWYWGQGT QVTVSSGGGE 120 CS 122 Sequence Number (ID): 64 Length: 122 Molecule Type: AA Features Location/Qualifiers: - source, 1..122 > mol_type, protein > note, ABT502. VHH nanobody variant > organism, synthetic construct Residues: QVQLQDSGGG LVQAGGSLRL SCEASGSIFS ISAMGWYRQA PGKQRELVAV ITTDGSTNYA 60 DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT DVYYCGASPR ALKWYWGQGT QVTVSSGGGG 120 SC 122

195 30123-WO-PCT

Claims

CLAIMS 1. A composition of matter comprising: an IgG4-binding moiety, a cellular receptor-binding moiety capable of binding to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors which are on surface degrading cells, and a linker moiety connecting the IgG4-binding moiety and the cellular receptor-binding moiety.

2. The composition of matter of claim 1, having a structure of: R^CN−(Xaa)y−R^CC, or a salt thereof. 3. The composition of matter of claim 1, wherein the composition of matter is selected from the group consisting of AGN401, AGN402, AGN403, AGN404, AGN501, AGN502, AGN508, and AGN509.

196 30123-WO-PCT

4. The composition of matter of claim 1, wherein the IgG4-binding moiety is an anti-IgG4 antibody or an antigen-binding fragment thereof.

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

6 The composition of matter of claim 1, wherein the IgG4-binding moiety comprises three regions having the structures of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

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

8. The composition of matter of claim 1, wherein the cellular receptor-binding moiety has the structure: ; groups, preferably R^A is a methyl or ethyl group optionally substituted with from 1-3 fluoro groups; Z_A is -(CH₂)_IM, -O-(CH₂)_IM, S-(CH₂)_IM, NR_M-(CH₂)_IM, C(O)-(CH₂)_IM-, a PEG group containing from 1 to 8 preferably 1-4 ethylene glycol residues or a -C(O)(CH₂)_IMNR_M group (preferably a PEG containing group comprising from 1 to 8 ethylene glycol, preferably 2-4 ethylene glycol residues); and Z_B is absent, (CH₂)_IM, C(O)-(CH₂)_IM- or C(O)-(CH₂)_IM-NR_M,.

9. The composition of matter of claim 1, wherein the cellular receptor-binding moiety is selected from the group consisting of TBT103, TBT104, TBT105, and TBT106.

197 30123-WO-PCT

10. The composition of matter of claim 1, for use as a medicament.

11. The composition of matter of claim 1, for use in removing IgG4 from a subject.

12. The composition of matter of claim 1, for use in treating a disease state or condition associated with the upregulation of IgG4 in a subject or patient.

13. The composition of matter of claim 1, for use in treating an IgG4-related disease in a subject or patient.

14. A method of making the composition of matter of claims 1-9, wherein the method comprises a conjugation step to link an IgG4-binding moiety to a cellular receptor-binding moiety through a linker moiety, wherein the conjugation step is selected from the group consisting of a MATE conjugation step, a maleimide conjugation step, and a sortase conjugation step.

15. A pharmaceutical composition comprising the composition of matter of claims 1-9 and a pharmaceutically acceptable excipient.

16. A method of removing IgG4 from a subject or patient comprising administering to the subject or patient the composition of matter of any of claims 1-9.

17. A method of treating a disease state or condition associated with the upregulation of IgG4 in a subject or patient comprising administering to the subject or patient an effective amount of the composition of matter of any of claims 1-9.

18. A method of treating an IgG4-related disease in a subject or patient comprising administering to the subject or patient an effective amount of the composition of matter of any of claims 1-9.

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19. A composition comprising: a first composition of matter comprising: an IgG4-binding moiety, a cellular receptor-binding moiety capable of binding to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors which are on surface degrading cells, and a linker moiety connecting the IgG4-binding moiety and the cellular receptor-binding moiety, and at least one additional composition of matter comprising a moiety comprising: a cellular receptor-binding moiety capable of binding to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors which are on surface degrading cells, and a linker moiety connecting the IgG4-binding moiety and the cellular receptor-binding moiety,

20. The composition of 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 a combination thereof.

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