TNF receptor superfamily agonist fusion polypeptides

Abstract

The present disclosure provides TNF receptor superfamily agonist fusion polypeptides having one or more metal hydroxide-binding polypeptides, complexes of the fusion proteins with metal hydroxides, compositions of fusion proteins and complexes thereof, and methods of preparing and using the same, e.g., to treat diseases such as cancer or autoimmune disease.

Description

Attorney Docket: AN K-005PC / 138388-5005TNF RECEPTOR SUPERFAMILY AGONIST FUSION POLYPEPTIDESFIELD

[0001] The present disclosure relates to, in part, fusion proteins comprising immunomodulatory moieties and a metal binding polypeptides, including metal complexes thereof, and methods of preparing and using the same, e.g., to treat diseases.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 718,204, filed November 8, 2024, the entire contents are incorporated by reference.SEQUENCE LISTING

[0003] The instant application contains a sequence listing, which has been submitted in XML format via Patentcenter. The XML file is named “138388-5005_Seq uence_Listi ng xml, ” which was created on November 5, 2025, and is approximately 458,752 bytes in size, the entire contents of which are incorporated herein by reference in their entirety.BACKGROUND

[0004] Various TNF receptor superfamily (TNFRSF) agonists have shown promise in the treatment of cancer. However, these treatments continue to face significant challenges, including dose-limiting toxicities, for example as described in Milling et al., “Delivering safer immunotherapies for cancer,” Adv. Drug. Deliv. Rec. (2017) Vol. 114, No. 79; Dadas et al., “Delivering co-stimulatory tumor necrosis factor receptor agonism for cancer immunotherapy: past, current and future perspectives,” Front. Immunol. (2023) Vol. 14; and Li and Wang, “Characteristics and clinical trial results of agonistic anti-CD40 antibodies in the treatment of malignancies,” Oncol. Lett. (2020) Vol. 20, No. 176). Indeed, many reports have described an unhelpful inverse correlation between efficacy and safety. For example, CD27 agonist antibodies have been shown to produce only partial response in some patients (see, for example, Burris et al., “A phase I study of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies or solid tumors,” J Immunother Cancer. (2013) 1 (Suppl 1 ):P127). Similarly, 4-1 BB agonist antibody, urelumab, induces significant liver toxicity in cancer patients (Segal et al., “Results from an Integrated Safety Analysis of Urelumab, an Agonist Anti-CD137 Monoclonal Antibody,” Clin Cancer Res. (2017) Vol. 23, No. 8, pp: 1929-36), whereas a more tolerable alternative, utomilumab, is yet to induce robust clinical efficacy with still-lingering toxicitiesPage 1 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005(Segal et al., “Phase I Study of Single-Agent Utomilumab (PF-05082566), a 4-1 BB / CD137 Agonist, in Patients with Advanced Cancer,” Clin Cancer Res. (2018) Vol. 24, No. 8, pp: 1816-23).

[0005] Similar correlations between efficacy and toxicity have been observed with agonists targeting other TNFRSF polypeptides. Agonist antibodies targeting 0X40, GITR, and TNFR2 have not produced significant benefit when used as monotherapies (e.g., Diab et al. Immunother Cancer. 27(6). 2016, Geva et al. Cancer. 126(22). 2020 and Heinhuis et al. JAMA Oncol. 6(1). 2020); combinations with checkpoint inhibitors improved efficacy but induced moderate to severe adverse events in 25-50% of patients receiving them (see, for example, Goldman et al. J Clin Oncol. 38(15). 2020 and Geva et al. Cancer. 126(22). 2020).

[0006] CD40 receptor agonists have been shown to produce substantial antineoplastic effects in several tumor models in vivo and several potential CD40 agonists, including both antibody and non-antibody agents, have been produced and investigated in clinical trials. Unfortunately, significant adverse events have been reported from such trials. For example, reports from early clinical trials with certain CD40 agonist antibodies describe adverse events such as cytokine release syndrome (also known as “cytokine storm”), hepatotoxicity, thromboembolic events, and sustained T-cell margination associated with repeated antibody dosing. A nonantibody CD40 agonist, MEDI-5083, produced severe toxicity in patients, resulting in one patient death. Studies with fewer adverse events have unfortunately shown limited efficacy (Vonderheide et al. J Clin Oncol. 25(7). 2007 and de Vos et al. J Hematol Oncol. 12:7:44. 2014).

[0007] Agonistic TNFRSF agents offer a therapeutic option which has the potential to generate anticancer immunity and / or autoimmune disease treatment by various mechanisms. CD40, for example, is a tumor necrosis factor receptor superfamily member expressed broadly on antigen-presenting cells (APC) such as dendritic cells (DCs), B cells, and monocytes, as well as many non-immune cells and a range of tumors. Agonistic CD40 agents are fundamentally different from agents which block negative immune checkpoint such as anti-CTLA-4 or anti-PD-1 antibodies. Despite initial clinical trials of agonistic CD40 / CD40L agents showing somewhat promising results, none have reached clinical approval yet.

[0008] The development of agonistic TNFRSF agents as a novel therapy has not been without its challenges. Adverse events associated with targeting numerous family members including at least CD40, CD27, 4-1 BB, 0X40, GITR, and TNFR2 signaling axes, including the prospect of triggering cytokine release syndromes, autoimmune reactions, thromboembolic syndromes (e.g., because CD40 is expressed by platelets and endothelial cells), hyper immune stimulation leading to activation-induced cell death or tolerance, and tumor angiogenesis possibly on the basis of CD40-dependent activation of tumor endothelial cells. The most Page 2 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 common side effects include cytokine release syndrome, resulting in chills, fever, rigors, and other symptoms soon after infusion. Agonist CD40 agents, for example, have also triggered mild elevations in liver enzymes and decreases in circulating platelet numbers.

[0009] While toxicity associated with TNFRSF agonists might be manageable using rigorous clinical intervention, there are opportunities to improve the clinical drawbacks of each. There remains a need to address the shortcomings of once-promising anti-cancer targets and to develop agonists, e.g., for use in clinically-relevant systems.SUMMARY

[0010] Accordingly, the present disclosure provides, tumor necrosis factor receptor superfamily (TNFRSF) polypeptide agonists fused with metal hydroxide-binding polypeptides useful for improving safety and efficacy of treatments. In embodiments, the metal hydroxide-binding polypeptides is phosphorylated and useful for anchoring / complexing the agonist with a metal hydroxide, such as aluminum hydroxide (e.g., alum). In embodiments, phosphorylation and / or metal hydroxide anchoring restricts localization of the agonist to a tumor environment (e.g., solid tumor environment). In embodiments, the agonists herein still exhibit abscopal effects despite fusion with a metal hydroxide-binding polypeptide and / or phosphorylation state.

[0011] In embodiments, aluminum binding technologies are particularly effective for achieving restricted localization, and improvement treatments by dampening toxicities associated with TNFRSF agonists, such as CD40 (e.g., improving safety and / or efficacy), enabling their use as an agonist therapy for cancer and / or autoimmune disease.

[0012] In embodiments, the metal binding moieties (e.g., metal hydroxide-binding polypeptides) and aluminum hydroxide (aka AI(OH)3 binding moieties) have been identified, for example, as described in WO2020263399, WO2022235755, WO2024102400 and WO2024102387, each of which is incorporated herein by reference in their entirety. In embodiments, the present disclosure provides an insight that fusion with such metal binding moieties (specifically, metal hydroxide binding moieties and in particular aluminum hydroxide binding moieties) are particularly useful when applied to TNFRSF polypeptide agonists, such as CD40 agonists.

[0013] In some embodiments, a TNFRSF polypeptide agonist for use in accordance with the present disclosure is or comprises an antibody that binds specifically to the TNFRSF polypeptide. In some embodiments, a TNFRSF polypeptide agonist utilized in accordance with the present invention is or comprisesPage 3 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 an antibody agent that binds specifically to the TNF receptor; in particular, embodiments, such antibody agent is a single chain antibody agent (e.g., a scFv, a camelid antibody, a diabody, another single-chain antibody format, etc). In some embodiments, a TNFRSF polypeptide agonist for use in accordance with the present disclosure is or comprises a TNFRSF ligand (e.g., a natural ligand, a modified natural ligand, an engineered ligand etc.).

[0014] In some embodiments, a TNFRSF polypeptide agonist is or comprises an agonist of one or more TNFRSF polypeptides selected from the group consisting of CD40 (p50 or TNFRSF5), TNFR1 (CD120a or TNFRSF1A), TNFR2 (CD120b or TNFRSF1A), LTBR (CD18 or TNFRSF3), 0X40 (CD134 or TNFRSF4), CD27 (Tp55 or TNFRSF7), CD30 (Ki-1 or TNFRSF8), FasR (APO-1, APT, CD95 or TNFRSF6), Dcr1 (TRAILR3, CD263 or TNFRSF10C), Dcr2 (TRAILR4, CD264 or TNFRSF10D), Dcr3 (TR6, M68 or TNFRSF6B7), DR3 (Apo-3, LARD, TRAMP or TNFRSF25), DR4 (APO-2, TRAILR1, CD261 or TNFRSFWA), DR5 (TRAILR2, CD262 or TNFRSFWB), DR6 (CD358 or TNFRSF21), 4-1 BB (CD137 or TNFRSF9), RANK (TRANCER, CD265 orTNFRSFUA), OPG (OCIF or TNFRSFU B), TWEAKR (CD266 orTNFRSF12A), TACI (CD267 or TNFRSF13B), BAFF-R (or CD268 or TNFRSF13C), HVEM (CD270 or TNFRSF14), LNGFR (P75NTR, NGFR or CD271), BCMA (CD269 or TNFRSF17), GITR (AITR, CD357, orTNFRSF18), TROY (TAJ, TRADE, or TNFRSF19), and EDA2R (TNFRSF27); in some such embodiments, such agonist is or comprises an antibody agent, or is or comprises a ligand (e.g., a natural ligand, a modified natural ligand, an engineered ligand etc.) that specifically binds to such TNFRSF polypeptide. The present disclosure provides particular entities in which an agonist of such a TNFRSF polypeptide is associated (e.g., linked covalently with, such as in a conjugate or a fusion protein with a metal-binding moiety (e.g., a metal-hydroxide binding moiety, for example specifically an aluminum hydroxide binding moiety) as described herein.

[0015] Without wishing to be bound by any particular theory, the present disclosure proposes that association (e.g., electrostatics, ionic interactions, Van der Waals forces, hydrogen bonding, etc.) with a metalbinding moiety (e.g., a metal-hydroxide binding polypeptide, for example, for aluminum hydroxide) as described herein may be particularly beneficial for TNFRSF agonists, particularly for TNFRSF agonists with safety, toxicity, dose-limiting, and efficacy issues. In embodiments, the present disclosure proposes that such association allows the relevant agonist entity to complex with a metal (e.g., aluminum), specifically in the form of a metal hydroxide (e.g., aluminum hydroxide), and that such complexation can retain and / or otherwise localize the agonist entity, such that certain potential undesired effects (e.g., with potential undesired safety consequences) are reduced. Alternatively, or additionally, in some embodiments, such complexation mayPage 4 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 improve efficacy of a particular agonist (e.g., relative to its uncomplexed form and / or a version that is not associated with the metal binding moiety). In some embodiments, a metal-hydroxide (e.g., aluminum hydroxide) form of an agonist entity as described herein shows superior potency than does an appropriate reference (e.g., a non-complexed version of the same agonist entity and / or the same base agonist not associated with the relevant, or, in some embodiments, any, metal binding moiety as described herein.

[0016] Various immunomodulatory polypeptides have shown significant promise in the treatment of cancer, but significant challenges remain, including dose-limiting toxicities (see, for example, Milling et al., Adv. Drug. Deliv. Rec. 114:79, 2017, Dadas et al. Front. Immunol. 14, 2023 and Li and Wang, Oncol. Lett. 20: 176, 2020,). Indeed, many reports have described an unhelpful inverse correlation between efficacy and safety. For example, CD27 agonist antibodies have been shown to produce only partial response in patients when tested as monotherapies for treating cancer (see, for example, Burris et al. J Immunother Cancer. P127. 2013). However, combination therapies (e.g., with rituximab or nivolumab) that improve efficacy produce moderate to severe adverse events in patients (as shown, for example, in Sanborn et al. J Immunother Cancer. 10(8):e005147. 2022 and Lim et al. Blood. 138 (Supplement 1): 715. 2021). Similarly, 4-1 BB agonist antibody, urelumab, induces significant liver toxicity in cancer patients (Segal et al. Clin Cancer Res. 23(8). 2017), whereas a more tolerable alternative, utomilumab, is yet to induce robust clinical efficacy (Segal et al. Clin Cancer Res. 24(8). 2018).

[0017] CD40 receptor agonists have been shown to produce substantial antineoplastic effects in several tumor models in vivo and several potential CD40 agonists, including both antibody and non-antibody agents, have been produced and investigated in clinical trials. Unfortunately, significant adverse events have been reported from such trials. For example, reports from early clinical trials with certain CD40 agonist antibodies describe adverse events such as cytokine release syndrome (also known as “cytokine storm”), hepatotoxicity, thromboembolic events, and sustained T-cell margination associated with repeated antibody dosing. A nonantibody CD40 agonist, MEDI-5083, produced severe toxicity in patients, resulting in one patient death. Studies with fewer adverse events have unfortunately shown limited efficacy (Vonderheide et al. J Clin Oncol. 25(7). 2007 and de Vos et al. J Hematol Oncol. 12:7:44. 2014).

[0018] Similar correlations between efficacy and toxicity have been observed with agonists targeting other TNFRSF polypeptides. For example, agonist antibodies targeting 0X40, GITR or TNFR2 have not produced significant benefit when used as monotherapies (e.g., Diab et al. Immunother Cancer. 27(6). 2016, Geva et al. Cancer. 126(22). 2020 and Heinhuis et al. JAMA Oncol. 6(1 ). 2020); combinations with checkpointPage 5 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 inhibitors improved efficacy but induced moderate to severe adverse events in 25-50% of patients receiving them (see, for example, Goldman et al. J Clin Oncol. 38(15). 2020 and Geva et al. Cancer. 126(22). 2020).

[0019] Recent work, as described for example, in International Patent Applications WO2020263399, WO2022235755, WO2024102400 and WO2024102387 has described fusion of certain immunomodulatory polypeptides with polypeptides characterized by an ability to complex with metal hydroxides. In embodiments, fusion with the metal hydroxide-binding polypeptide acts as a particulate scaffold able to persist at the site of administration (e.g., injection) for extended periods of time (e.g., days to weeks), and that such complexation (i.e., association of the fusion polypeptide with the metal hydroxide) retains the fusion polypeptide (including its immunomodulatory polypeptide moiety) at the injection site (e.g., intratumoral injection) as a depot. Accordingly, in embodiments, the immunomodulatory polypeptide persists within the tumor microenvironment, thereby enhancing efficacy, and systemic exposure is limited, thereby reducing toxicity.

[0020] WO2020263399 demonstrated that polypeptides amenable to phosphorylation can adsorb to aluminum hydroxide {e.g., also referred to herein as “alum”) when in their phosphorylated form (i.e., through ligand-exchange reactions where phosphate groups in the polypeptide replace hydroxyl groups in the aluminum hydroxide). In embodiments, various alum-binding peptides (“ABPs”) are developed that include phosphorylation sites for the Fam20C kinase and that are fused with agonists (e.g., immunomodulatory polypeptides). In embodiments, fusion polypeptides are generated, in which an alum-binding polypeptide was linked to the N- or C- terminus of an immunomodulatory polypeptide, typically by way of a linker (e.g., amino acid linker). In embodiments, the extent of phosphorylation of these fusion polypeptides is assessed when expressed alone or in combination with Fam20C kinase.

[0021] In embodiments, the TNFRSF agonist agent is a CD40 agonist fused with a metal hydroxide- binding polypeptide. In embodiments, the CD40 agonist is phosphorylated but not part of a metal hydroxide complex e.g., is capable of efficacy without anchoring to a metal hydroxide). Alternatively, in embodiments, CD40 agonists herein are anchored to a metal hydroxide. In embodiments, results reported herein document surprising effectiveness of these strategies, and provide engineered CD40 agonists with surprising and beneficial attributes. In embodiments, described herein are metal-hydroxide binding polypeptides characterized by improved CD40 signaling potency with and without adsorption to aluminum hydroxide (e.g., ABP20-G4-GE or ABP20-G4-8x-GE).

[0022] In embodiments, the present disclosure provides particularly useful fusion polypeptides (e.g., as shown in Figure 1). In embodiments, the present disclosure teaches effectiveness of the fusion polypeptides Page 6 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 in treating a subject with a tumor or autoimmune disease. In embodiments, the agent is administered as a monotherapy. In embodiments, the agent is administered in combination, for example, with immune-modulator therapy, chemotherapeutic agent, or surgical resection.

[0023] In embodiments, described herein are methods for producing fusion polypeptides herein (e.g., CD40 agonists). In embodiments, described herein are compositions (e.g., pharmaceutical compositions) of fusion polypeptides herein (e.g., CD40 agonists). In embodiments, provided technologies achieve reproducible production of fusion peptide preparations, including specifically phosphorylated preparations. In embodiments, provided technologies may include, for example, expression, purification, and / or analytical technologies. In embodiments, the present disclosure provides desirable preparations of provided fusion polypeptides, including in some embodiments phosphorylated preparations and / or in some embodiments, preparations of fusion polypeptides (e.g., phosphorylated fusion polypeptides) complexed with a metal hydroxide.

[0024] In embodiments, the present disclosure provides fusion polypeptides comprising: (a) an immunomodulatory polypeptide that comprises a TNFRSF polypeptide agonist moiety (e.g., CD40); and (b) a metal-hydroxide binding polypeptide comprising an amino acid sequence having a plurality of phosphorylation sites. In embodiments, the fusion protein adopts phosphorylated and unphosphorylated forms. In embodiments, the fusion polypeptide, when exposed to a metal hydroxide, forms a complex therewith. In embodiments, the metal hydroxide is aluminum hydroxide. In embodiments, the complex forms more readily when the metal-hydroxide-binding polypeptide in a phosphorylated form than when it is in an unphosphorylated form.

[0025] In embodiments, one or more of the phosphorylation sites is targeted by a Fam20C kinase. In embodiments, the phosphorylation site is or comprises an S-X-E motif. In embodiments, the plurality of phosphorylation sites comprises 4 or more S-X-E motifs. In embodiments, the plurality of phosphorylation sites comprises 8 or more S-X-E motifs.

[0026] In embodiments, at least two adjacent S-X-E motifs are separated by a spacer. In embodiments, the spacer comprises at least one glycine residue. In embodiments, the spacer comprises a plurality of glycine residues. In embodiments, the spacer comprises at least four glycine residues. In embodiments, the spacer has a sequence that comprises four glycine residues. In embodiments, each SXE motif is separated from each adjacent S-X-E motif by a spacer.

[0027] In embodiments, the present disclosure provides method of treating a subject with a tumor, the method comprising a step of: treating the subject with (i) a CD40 agonist with a metal hydroxide-binding Page 7 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 polypeptide without a metal hydroxide, or with (ii) a complex comprising: (a) a CD40 agonist with a metal hydroxide-binding polypeptide and (b) a metal hydroxide. In embodiments, (a) and (b) are formulated together. In embodiments, (a) and (b) are mixed prior to administration, or substantially simultaneously with administration.

[0028] In embodiments, the complex is administered by intratumoral injection. In embodiments, the complex is administered by peritumoral injection. In embodiments, the complex is administered to a tumordraining lymph node or lymph nodes.

[0029] In embodiments, the complex is administered in combination with a second therapeutic. In embodiments, the second therapeutic is radiation. In embodiments, the second therapeutic is surgical tumor resection. In embodiments, the fusion polypeptide is administered prior to surgical tumor resection. In embodiments, the second therapeutic is a chemotherapy. In embodiments, the second therapeutic is an antitumor antibody. In embodiments, the second therapeutic is a targeted therapy (e.g., BRAF inhibitor, MEK inhibitor, etc.). In embodiments, the second therapeutic is an immune modulator. In embodiments, the immune modulator is a checkpoint inhibitor. In embodiments, the checkpoint inhibitor is an antibody or a functional fragment thereof. In embodiments, the antibody targets one or more of PD-1, PD-L1 , CTLA-4, TIM3, TIGIT, and LAG3. In embodiments, the antibody targets PD-1. In some embodiments, the antibody comprises a tumortargeting CD3 bispecific antibody or antibody agent. In embodiments, the immune modulator is a cell therapy. In some embodiments, the cell therapy is selected from the group consisting of: CAR-T cells, TCR-T cells, ex- vivo expanded TILs, and NK cells.

[0030] In embodiments, the present disclosure provides methods of treating a subject with a tumor comprising administering a fusion polypeptide comprising: treating the subject with (i) a CD40 agonist with a metal hydroxide-binding polypeptide without a metal hydroxide, or with (ii) a complex comprising: (a) a CD40 agonist with a metal hydroxide-binding polypeptide and (b) a metal hydroxide, where the subject has received or is receiving therapy with at least one additional therapeutic. In embodiments, the CD40 agonist and metal- hydroxide are formulated together, forming a complex. In embodiments, the CD40 agonist and metal-hydroxide are mixed prior to administration, or substantially simultaneously with administration.

[0031] In embodiments, the CD40 agonist is administered by intratumoral injection. In embodiments, the CD40 agonist is administered by peritumoral injection. In embodiments, the CD40 agonist is administered to a tumor-draining lymph node or lymph nodes. In embodiments, the CD40 agonist is not complexed with a metal hydroxide and is suitable for systemic administration.Page 8 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0032] In embodiments, the immune agonist moiety of a fusion polypeptide disclosed herein comprises a moiety or a functional fragment thereof. In embodiments, the functional fragment is signaling competent. In embodiments, the moiety comprises a TNFRSF polypeptide agonist moiety or a functional fragment thereof. In embodiments, the TNFRSF polypeptide agonist moiety comprises a TNFRSF polypeptide agonist antibody or an antibody agent. In embodiments, the TNFRSF polypeptide agonist moiety comprises a TNFRSF polypeptide agonist ligand.

[0033] In embodiments, a TNSRSF polypeptide agonist is an antibody or an antibody agent. In embodiments, the TNFRSF polypeptide antibody or an antibody agent is a CD27 agonist antibody or an antibody agent, a 4-1 BB agonist antibody or an antibody agent, a CD40 agonist antibody or an antibody agent, a 0X40 agonist antibody or an antibody agent, a GITR agonist antibody or an antibody agent or a TNFR2 agonist antibody. In some embodiments, the TNFRSF polypeptide agonist antibody or an antibody agent is a CD40 agonist antibody. In some embodiments, the CD40 antibody or antibody agent comprises a heavy chain (HC) and a light chain (LC). In some embodiments, the CD40 antibody agent is or comprises a single-chain variable fragment (scFv). In some embodiments, the CD40 antibody agent is or comprises a diabody (e.g., a non-covalent dimer of single-chain scFv fragments connected by a peptide linker In some embodiments, the CD40 antibody agent is or comprises a single-chain (Fv)2 in which two scFv fragments are covalently linked). In some embodiments, the CD40 antibody agent is or comprises a single-domain, camelid-derived VHH antibody. In some embodiments, a CD40 agonist antibody or antibody agent binds specifically to CD40 expressed by: a) dendritic cells; b) B cells; c) macrophages; d) cancer cells, or a combination thereof.

[0034] In embodiments, the TNFRSF polypeptide agonist moiety comprises a TNFRSF polypeptide agonist ligand. In some embodiments, the TNFRSF polypeptide agonist moiety comprises a TNFRSF polypeptide agonist fusion polypeptide. In some embodiments, the TNFRSF polypeptide agonist fusion polypeptide comprises a first and a second moiety. In some embodiments, the first moiety comprises a TNFRSF polypeptide agonist ligand. In some embodiments, the first moiety comprises an immunoglobulin G (IgG). In some embodiments, the first moiety comprises a T4 fibritin foldon domain (T4D). In some embodiments, the first moiety comprises a modified GCN4-derived leucine zipper (GCN4). In some embodiments, the second moiety comprises a TNFRSF polypeptide agonist ligand. In some embodiments, the second moiety comprises an immunoglobulin G (IgG). In some embodiments, the second moiety comprises a T4 fibritin foldon domain (T4D). In some embodiments, the second moiety comprises a modified GCN4-derived leucine zipper (GCN4). In some embodiments, the first and second moieties or functional fragments thereofPage 9 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 are linked via a first linker. In some embodiments, the first linker comprises a polypeptide. In some embodiments, the polypeptide comprises a (G4S) linker. In some embodiments, the polypeptide comprises a (G4S)2 linker. In some embodiments, the polypeptide comprises a (G4S)3 linker. In some embodiments, the polypeptide comprises a (G4S)4 linker.

[0035] In some embodiments, the first or the second moiety comprises a TNFRSF polypeptide agonist ligand. In some embodiments, the TNFRSF polypeptide agonist ligand is selected from CD27L, 4-1 BBL, CD40L, OX40L, GITRL, membrane TNF, D18, TNF-a, and LT .

[0036] In some embodiments, the first or the second moiety comprises an immunoglobulin G (IgG) or a functional moiety thereof. In some embodiments, the functional moiety is an IgG Fc. In some embodiments, the IgG Fc is human lgG1 Fc. In some embodiments, the IgG Fc is human lgG2 Fc. In some embodiments, the IgG Fc is human lgG4 Fc. In some embodiments, the IgG Fc further comprises a hinge region of an IgG.

[0037] In some embodiments, the first or the second moiety comprises one or more, two or more, or three or more TNFRSF polypeptide ligands. In some embodiments, the first or the second moiety comprises one or more, two or more, or three or more CD40L polypeptides. In some embodiments, two CD40L polypeptides are linked together with a linker, wherein a first CD40L polypeptide is at the 5’ end is linked to the 3’ end of the second CD40L. In some embodiments, three CD40L polypeptides are linked together with a linker, wherein the first CD40L polypeptide is at the 3’ end is linked to the 5’ end of the second CD40L, which is also linked at the 3’ end to the 5’ end of the third CD40L polypeptide.

[0038] In some embodiments, an immunomodulatory polypeptide of a fusion polypeptide disclosed herein and a metal-hydroxide binding polypeptide are linked via a second linker. In some embodiments, the second linker comprises a polypeptide. In some embodiments, the polypeptide comprises the amino acid sequence, GGGGE. In some embodiments, the polypeptide comprises the amino acid sequence, GGGGEGGGG. In some embodiments, the polypeptide comprises the amino acid sequence, GGGGS. In some embodiments, the polypeptide comprises the amino acid sequence, GGGGSGGGG. In some embodiments, the metal- hydroxide binding polypeptide is linked directly to the c-terminus of the immunomodulatory polypeptide. In some embodiments, the metal-hydroxide binding polypeptide is linked via a second linker to the c-terminus of the immunomodulatory polypeptide.

[0039] In some embodiments, the present disclosure provides a method of manufacturing a phosphorylated form of fusion polypeptides disclosed herein by contacting the fusion polypeptide with a kinase. In some embodiments, contacting comprises co-expressing the fusion polypeptide and a kinase. In some Page 10 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 embodiments, the fusion polypeptide and kinase are co-expressed at a ratio of 2:1 to 100:1. In some embodiments, the ratio is 4:1. In some embodiments, the 4:1 ratio is achieved using two separate plasmids to express the fusion polypeptide and the kinase. In some embodiments, the two separate plasmids comprise promoters of differing strength to produce the ratio of 4:1. In some embodiments, the ratio is 8:1. In some embodiments, the 8:1 ratio is achieved using a single vector with two promoters to express the fusion polypeptide and the kinase. In some embodiments, the kinase is Fam20C.

[0040] In some embodiments, the step of co-expressing comprises expressing from promoters established to direct expression at the ratio. In some embodiments, the step of co-expressing comprises expressing from a bicistronic construct.

[0041] In some embodiments, the method further comprises a step of purifying the phosphorylated form. In some embodiments, the step of purifying comprises affinity chromatography.

[0042] In some embodiments, the fusion polypeptide is exposed to a metal-hydroxide to form a complex therewith. In some embodiments, the complex is prepared prior to administering to a subject. In some embodiments, a fusion polypeptide of the present disclosure is manufactured by contacting a phosphorylated form of a fusion polypeptide with a metal hydroxide.

[0043] In some embodiments, a complex comprises a fusion polypeptide of the present disclosure and a metal hydroxide. In some embodiments, the complex comprises an average of 2-8 phosphates per fusion polypeptide. In some embodiments, the complex is characterized as having greater than 95% metal hydroxide retention. In some embodiments, the complex comprises a ratio of 1:1 to 1 :20 by mass of fusion polypeptide to metal hydroxide, e.g., as defined by metal mass. In some embodiments, the ratio is 1 :5 to 1 :20 by mass of fusion polypeptide to metal hydroxide, e.g., as defined by metal mass. In some embodiments, the ratio is 1 :10 by mass of fusion polypeptide to metal hydroxide, e.g., as defined by metal mass. In some embodiments, the ratio is 1 :5 by mass of fusion polypeptide to metal hydroxide, e.g., as defined by metal mass.

[0044] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a fusion polypeptide as disclosed herein. In some embodiments, a pharmaceutical composition is formulated as a fusion polypeptide metal-hydroxide complex.

[0045] In some embodiments, the present disclosure provides methods of characterizing a preparation of a fusion polypeptide of the present disclosure by assessing degree of phosphorylation. In some embodiments, the degree of phosphorylation is assessed by determining the number of phosphates perPage 11 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 protein. In some embodiments, the number of phosphates per protein is determined using a colorimetric assay. In some embodiments, the colorimetric assay is a malachite green assay.

[0046] In some embodiments, the present disclosure provides methods of characterizing a fusion polypeptide of the present disclosure by assessing signaling activity of the fusion polypeptide. In some embodiments, the signaling activity is assessed in vitro. In some embodiments, in vitro assessment utilizes a reporter assay.

[0047] In some embodiments, the present disclosure provides methods of characterizing a fusion polypeptide of the present disclosure by assessing phosphate content. In some embodiments, assessing phosphate content comprises use of a malachite green assay. In some embodiments, assessing phosphate content comprises use of a high performance liquid chromatography assay. In some embodiments, high performance liquid chromatography assay utilizes a SAX-10 column.

[0048] In some embodiments, the present disclosure provides methods of characterizing a fusion polypeptide of the present disclosure by assessing potency of the preparation. In some embodiments, potency is characterized by assessing immune moiety signaling. In some embodiments, immune moiety signaling is determined using a reporter assay. In some embodiments, potency is characterized by assessing TNFRSF polypeptide signaling. In some embodiments, potency is characterized by assessing CD40 signaling. In some embodiments, CD40 signaling is determined using a reporter assay. Additionally or alternatively, in some embodiments, potency is characterized by the binding strength of an anti-CD40 antibody or an antigen-binding fragment thereof to CD40.

[0049] In some embodiments, the present disclosure provides a method of characterizing a pharmaceutical composition as described herein comprising assessing one or more of: (a) the purity of the preparation; (b) phosphate content; (c) potency of the pharmaceutical composition; (d) efficacy of treating a subject having a tumor; and (e) combination with a second therapeutic agent.DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 provides an exemplary schematic of fusion polypeptide metal-hydroxide complexes of the present disclosure. Fusion polypeptide metal-hydroxide complexes can be administered to a subject and result in enhanced retention, safety, and / or efficacy compared to an appropriate reference standard.

[0051] Figures 2A-2D provides a non-limiting diagrammatic representation of illustrative fusion polypeptides of the present disclosure comprising a CD40 agonist antibody or antibody agent and a metal-Page 12 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 hydroxide binding polypeptide with a plurality of phosphorylation sites. Figure 2A depicts various antibodyformat molecules for anti-CD40 metal hydroxide complexes. Figure 2B depicts an illustrative metal hydroxide adsorption method. Figures 2C and 2D depicts a metal hydroxide retention data showing anti-CD40 antibody retention and the use of mouse serum and / or free phosphate to liberate adsorbed protein.

[0052] Figure 3 provides a diagram of an exemplary fusion polypeptide of the present disclosure comprising a CD40 agonist antibody or an antibody agent and a metal-hydroxide binding polypeptide with a plurality of phosphorylation sites activating dendritic cells (DCs) via a CD40 agonist function.

[0053] Figures 4A-4D show construct expression and purification of exemplary fusion polypeptides using sequential Ni-NTA affinity (for scFv constructs), protein A chromatography (Fc-containing constructs), and size exclusion chromatography (SEC). All polypeptides ran as a single peak on SEC, demonstrating there was no significant degradation or aggregation. Figure 4A shows results for CD40Ab1-ABP. Figure 4B shows results for CD40Ab1-ABP20-G4-GE. Figure 4C shows results for CD40Ab1-ABP20-G4-8x-GE. Figure 4D shows results for scFv1-ABP20-G4-8x-GE-His.

[0054] Figure 5 demonstrates analysis of phosphorylation levels using a malachite green assay of exemplary purified fusion polypeptides. All ABPs demonstrated significantly higher phosphorylation compared to negative controls (e.g., his tag-containing protein with no ABP sequence).

[0055] Figures 6A-6B demonstrate assessment of control CD40 ligand (CD40L), CD40Ab1, CD40AM- ABP20-G4-GE, CD40Ab1-ABP20-G4-8x-GE and scFv1-ABP20-G4-8x-GE in a CD40 reporter assay, wherein a luciferase reporter driven by a response element is expressed in response to CD40 agonist signaling. Figure 6A shows that the CD40L and CD40Ab1 without adsorption to aluminum hydroxide produced greater potency with respective EC50 values of 33.6 and 67.2 ng / mL than unadsorbed CD40Ab1-ABP20-G4-GE and CD40Ab1-ABP20-G4-8x-GE polypeptides with respective EC50 values of 216.0 ng / mL and 163.0 ng / mL, and the EC50 value for scFv1-ABP20-G4-8x-GE could not be obtained. Figure 6B shows that, when adsorbed to aluminum hydroxide, 7-9 times greater potency is exhibited by all ABP constructs compared to their unadsorbed counterparts. The EC50 values for CD40Ab1, CD40Ab1-ABP20-G4-GE, CD40Ab1-ABP20-G4- 8x-GE and scFv1-ABP20-G4-8x-GE were 65.2 ng / mL, 25.9 ng / mL, 18.7 ng / mL and 231.9 ng / mL, respectively. Alhydrogel® (aluminum hydroxide gel) was used as a negative control. All individual data points are means ± SD.

[0056] Figures 7A-7D demonstrate that anti-CD40-metal hydroxide-binding polypeptide fusions adsorbed to metal hydroxide (alum) activate primary human dendritic cells (DCs), but do not contribute to IL-6 Page 13 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 cytokine release. Figure 7A shows that the anti-CD40 fusion complexed with alum increased expression of CD86 compared to anti-CD40 fusion without alum and the anti-CD40 antibody alone. Figure 7B shows that the anti-CD40 fusion complexed with alum increased expression of CD80 compared to anti-CD40 fusion without alum. Figure 7C shows that the anti-CD40 fusion complexed with alum increased expression of IL-8 compared to anti-CD40 fusion without alum. Figure 7D shows that the anti-CD40 fusion complexed with alum and the anti-CD40 fusion without alum did not increase IL-6 expression as much as the anti-CD40 antibody alone.

[0057] Figure 8 depicts a diagrammatical representation of in vivo MC38 cancer trial for testing anti- CD40 fusions complexed with alum.

[0058] Figure 9 depicts a graphical representation of pharmacokinetic (PK) profile of anti-CD40 agonists herein from peripheral blood draws. Anti-CD40 fusion with alum anchoring resulted in about a 10-fold increase in tumor localization at approx. 1 week post-injection.

[0059] Figures 10A-10C depict graphical representations of the in vivo toxicity profile of anti-CD40 agonists herein. Figure 10A depicts serum measurements of liver enzyme, alanine aminotransferase (ALT) at various time points post-infection. Figure 10B depicts serum measurements of liver enzyme, aspartate aminotransferase (AST) at various time points post-injection. The acute increase in toxicity observed from anti- CD40 antibody alone is not observed with alum complexed anti-CD40 fusions. Figure 10C depicts that changes in body weight (as a proxy measure for toxicity) demonstrate that alum anchoring reduced the toxicity observed with the anti-CD40 antibody alone.

[0060] Figure 11 depicts a Kaplan Meier survival plot showing the in vivo survival benefits of anti-CD40 agonists herein. Each of the anti-CD40 antibody alone, metal hydroxide-binding polypeptide fusion, and alum complexed forms conferred a statistically significant improvement in overall survival (**p < 0.001 , log rank test). Number is as follows: 1 = vehicle; 2 = alum alone (lines overlap); 3 = CD40 mAb alone; 4 = CD40-ABP fusion; 5 = CD40-ABP fusion + alum.

[0061] Figure 12A-12B depicts graphical analysis of the effects on tumor volume (injected tumors) and abscopal effects (non-injected lesions). Figure 12A shows that each of the anti-CD40 antibody alone, metal hydroxide-binding polypeptide fusion, and alum complexed forms conferred a statistically significant reduction in tumor volume over time (**p < 0.001 , log rank test). Figure 12B shows that alum adsorption dampened the abscopal effect relative to the anti-CD40 antibody alone and non-complexed metal hydroxide-binding polypeptide fusion.Page 14 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0062] Figures 13A-13C depict graphical representations of tumor volume change as a function of time post-treatment in an MC38 colon cancer in vivo mouse model (expressing humanized CD40) due to treatment with exemplary CD40 agonists. The tumor volumes are from injected tumors in a dual flank tumor engraftment model. Figure 13A depicts tumor volume traces for treatment with alum vs. anti-CD40 antibody (non-fusion). Figure 13B depicts tumor volume traces for treatment with alum vs. anti-CD40-metal hydroxide-binding polypeptide agonist (anti-CD40-4x-ABP). Figure 13C depicts tumor volume traces for treatment with alum vs. anti-CD40-metal hydroxide-binding polypeptide agonist complexed with alum (anti-CD40-4x-ABP + alum).

[0063] Figures 14A-14C depict graphical representations of tumor volume change as a function of time post-treatment in an MC38 colon cancer in vivo mouse model (expressing humanized CD40) due to treatment with exemplary CD40 agonists. The tumor volumes are from the non-injected tumors in a dual flank tumor engraftment model demonstrating an abscopal effect. Figure 14A depicts tumor volume traces for treatment with alum vs. anti-CD40 antibody (non-fusion agonist). Figure 14B depicts tumor volume traces for treatment with alum vs. anti-CD40-metal hydroxide-binding polypeptide agonist (anti-CD40-4x-ABP). Figure 14C depicts tumor volume traces for treatment with alum vs. anti-CD40-metal hydroxide-binding polypeptide agonist complexed with alum (anti-CD40-4x-ABP + alum).

[0064] Figures 15A-15B depict graphical representations of composite curves (mean ± S.D.) for each in vivo group showing that agonists with the phosphorylated metal-hydroxide binding polypeptide did not adversely affect the activity of the anti-CD40 antibody, butthat alum anchoring worsened the efficacy compared to the anti-CD40 antibody alone. Figure 15 A shows tumor volumes for injected tumors. The anti-CD40 agonist without alum showed greatly increased efficacy via abscopal effects (in the non-injected tumor, Figure 15B).DETAILED DESCRIPTIONFusion polypeptide-metal hydroxide complexes

[0065] Immune-modulating therapeutic approaches can be useful in treating a variety of disease states, including cancer. Established immune-modulating therapeutic approaches include, but are not limited to, monoclonal antibodies, T cell therapy, immune checkpoint modulators, and immunomodulatory polypeptides. The present disclosure provides technologies that, among other things, include fusion polypeptides comprising an immunomodulatory polypeptide and a metal hydroxide binding polypeptide, as well as technologies to produce and / or utilize such fusion polypeptides (for example, Figure 1). In some embodiments, provided fusion polypeptides utilize a metal hydroxide binding polypeptide (e.g., aluminum hydroxide binding polypeptide) thatPage 15 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 displays one or more improved structural or functional characteristics relative to a reference polypeptide without a metal hydroxide binding polypeptide.

[0066] Among other things, the present disclosure provides a surprising discovery that improved (e.g., relative to a reference polypeptide without a metal hydroxide binding polypeptide) metal-hydroxide binding polypeptides can be developed. For example, in some embodiments, the present disclosure provides metal- hydroxide binding polypeptides characterized by signaling activity of polypeptides adsorbed to metal hydroxide (e.g., aluminum hydroxide) relative to an appropriate reference (e.g., to a polypeptide not adsorbed to alum, for example as described and / or exemplified herein). Alternatively or additionally, in some embodiments, provided metal-hydroxide binding polypeptides are characterized by improved intratumoral retention (e.g., after intratumoral injection) efficacy, as compared to such appropriate reference (e.g., alone and / or when included in a fusion polypeptide,).

[0067] In some embodiments, a fusion polypeptide in accordance with the present disclosure is characterized by improved tumor retention and / or efficacy as compared with an appropriate comparable reference fusion polypeptide that does not include a metal hydroxide binding polypeptide.

[0068] Among other things, the present disclosure provides particularly useful fusion polypeptides. Furthermore, the present disclosure teaches effectiveness of provided fusion polypeptides in treating a subject with a tumor. Still further, the present disclosure teaches that, in some embodiments, a particular fusion polypeptide is useful as a monotherapy and / or that, in some embodiments, a particular fusion polypeptide is particularly useful in combination therapy (e.g., in combination with an immune modulator such as an immune checkpoint inhibitor such as, for example, an anti-PD-1 agent such as an anti-PD-1 antibody.

[0069] Alternatively or additionally, in some embodiments, the present disclosure provides certain technologies for production and / or characterization of provided fusion polypeptides, compositions that comprise them, and / or components within them (e.g., immunomodulatory polypeptides, metal-hydroxide- binding polypeptides, linkers, etc.).

[0070] In some embodiments, provided technologies achieve reproducible production of fusion peptide preparations, specifically including phosphorylated preparations and / or metal-hydroxide-complexed preparations. In some embodiments, provided technologies may include, for example, expression, purification, and / or analytical technologies. Moreover, in some embodiments, the present disclosure provides desirable preparations of provided fusion polypeptides, including in some embodiments phosphorylated preparationsPage 16 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 and / or in some embodiments, preparations of fusion polypeptides (e.g., phosphorylated fusion polypeptides) complexed with a metal hydroxide.

[0071] In embodiments, described herein is a CD40 “agonist,” which is a protein or protein complex that comprises an anti-CD40 antibody that is capable of agonism through a CD40 / CD40L pathway. In embodiments, agonists herein are fused with one or more metal hydroxide-binding polypeptide (e.g., alum binding polypeptide, ABP). In embodiments, the one or more metal hydroxide-binding polypeptide is phosphorylated, or is configured to adopt a phosphorylated state via phosphorylation at one or more phosphorylation sites (e.g., by a kinase). In embodiments, agonists herein are not complexed to a metal hydroxide particle (e.g., such as alum). Without wishing to be bound by theory, agonists and herein demonstrated improved in vivo performance in comparison to cognate anti-CD40 agonist (not fused to metal hydroxide polypeptide), and improved in vivo performance in comparison to an anti-CD40 agonist (fused to metal hydroxide polypeptide) complexed with a metal hydroxide. In embodiments, the CD40 agonist, either with or without anchoring to alum, possess treatment improvements compared to an anti-CD40 antibody alone (without ABP fusion).

[0072] In embodiments, the CD40 agonist is anchored to alum, or alternatively not anchored to alum, depending upon treatment needs. Typically, metal hydroxide-binding polypeptides are used with the purpose of phosphorylating the peptide for complexing / anchoring (binding / associating) the polypeptide with a metal hydroxide (e.g., such as alum). In embodiments, agonists herein are substantially lacking and / or substantially uncomplexed with a metal hydroxide and / or metal. In embodiments, the “substantially lacking” and / or “substantially uncomplexed,” in reference to metal hydroxide and / or metal refers to a lack of metal hydroxide or metal particles complexed with the metal hydroxide-binding polypeptide. In embodiments, the metal hydroxide and / or metal comprises a particle has a dimension of less than about 1 nM to about or at least about 20 pm in size. In embodiments, this includes metal compounds larger than individual metal ions (e.g., larger than a metal salt ions, such as a sodium ion (Na+), magnesium ion (Mg2+), etc.). In embodiments, the “substantially lacking” and / or “substantially uncomplexed,” refers to a mass ratio of protein to metal hydroxide and / or metal of about or at least about 100:1 , about or at least about 500:1 , about or at least about 1 ,000:1 , about or at least about 5,000: 1 , about or at least about 10,000: 1 , about or at least about 50,000: 1 , or about or at least about 100,000:1 , including ratios therebetween. In embodiments, the mass ratio is a ratio in solution (e.g., at least about 100-times more protein mass in solution in comparison to the weight of any metals or metal hydroxide particles). In embodiments, CD40 agonists without metal hydroxide complexing are more suitablePage 17 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 for systemic administration, such as intravenous route administration, than a metal hydroxide complexed CD40 agonist.

[0073] In embodiments, “CD40” refers to “Cluster of Differentiation 40,” which is a costimulatory protein found on antigen-presenting cells that is required for their activation. In embodiments, the present disclosure provides CD40 binding molecules that specifically bind to and activate / agonize CD40 (e.g., CD40 agonists).Fusion polypeptides - Immunomodulatory polypeptide

[0074] The present disclosure provides an insight that it may be particularly useful to impart alum-binding capability to tumor necrosis factor receptor superfamily (TNFRSF) entities.

[0075] Among other things, the present disclosure appreciates that many potential utilities have been described for TNFRSF agonist entities, and significant investment has been made in efforts to develop therapeutic agents that are or include such entities. A non-limiting example of therapeutic agents developed as agonists of TNFRSF polypeptides comprise agonists of CD27, 4-1 BB, CD40, 0X40, GITR and TNFR2. Monotherapies with 4-1 BB, 0X40, GITR and TNFR2 antibody agonists have been moderately tolerated by patients, but they have not been clinically efficacious. On the other hand, putatively more efficacious combination therapies of TNFRSF polypeptide agonists with, for example, checkpoint inhibitors, is not well- tolerated by patients.

[0076] In embodiments, the agonist is or comprises an antibody that binds to one or more of the TNFRSF polypeptides. In embodiments, the agonist is or comprises a ligand that binds to one or more of the TNFRSF polypeptides (e.g., CD40L, etc.).

[0077] Present disclosure appreciates that one or more of these challenges may well be alleviated by successful design of an alum-binding fusion polypeptide which links a TNF receptor agonist entity with an alum binding domain. In embodiments, complexing / anchoring of monoclonal CD40 agonist antibodies to alum achieves more potent CD40 receptor signaling compared to non-alum bound CD40 agonist antibodies. In embodiments, alum-bound CD40 antibodies can be injected intratumorally, therefore curbing systemic toxicity and promoting immunomodulatory efficacy. Additionally or alternatively, in embodiments, alum-bound intratumorally injected CD40 agonist antibodies, if released inside a tumor from their alum binding, retain their signaling potency. Furthermore, in embodiments, alum binding of CD40 agonist antibodies may be desirable to achieve greater antibody affinity (Kd) to its antigen (e.g., CD40), greater rate of antibody association with its target antigen (kon), and / or lower rate of antibody dissociation from its target antigen (koff). Even further, inPage 18 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 embodiments, immunomodulatory moieties (e.g., CD40 agonists) may have greater potency when they are bound to alum comprising about or at least about 2 or more, about or at least about 4 or more, about or at least about 6 or more, or 8 or more S-X-E phosphorylation motifs, for example as demonstrated in Published PCT Application WO2022235755, the entire contents of which are incorporated by reference. In embodiments, alum binding of CD40 agonist antibodies administered intratumorally to a subject may improve overall survival when the CD40 agonist antibody is administered alone or in combination with other anti-neoplastic treatments (e.g., checkpoint inhibitors).

[0078] Without wishing to be bound by theory, fusions with metal hydroxide-binding polypeptides (with or without being complexed to a metal) facilitate penetration into the tumor microenvironment due to the concentration of negative charges provided by the phosphorylated metal hydroxide-binding polypeptides. In embodiments, the metal binding protein functions as a phosphate tag that alters one or more physicochemical properties of the overall fusion protein that enhances its properties as administrable agent, such as the overall surface charge (with phosphosrylation provided neg negative charge). In embodiments, complexing the fusion proteins with a metal hydroxide increases the local concentration of fusion protein, e.g., improving clustering and / or agonism of cell signaling.

[0079] Fusion polypeptides of the present disclosure comprise at least one immunomodulatory polypeptide.

[0080] In some embodiments, an immunomodulatory polypeptide is or comprises at least one immune agonist moiety. In some embodiments, an immune agonist moiety is or comprises a functional fragment of a parent (e.g., a wild type) polypeptide; for example, in some embodiments, an immunomodulatory polypeptide is or comprises a functional fragment that is a signaling competent fragment. In some embodiments, an immunomodulatory polypeptide comprises one, two, three, four, five, or six immune agonist moieties.

[0081] In some embodiments, a fusion polypeptide comprises two or more immunomodulatory polypeptides (e.g., two or more immune agonist moieties). In some such embodiments, a fusion polypeptide comprises two or more immunomodulatory polypeptides that are the same; in some such embodiments, all immunomodulatory polypeptides in a fusion polypeptide in accordance with the present disclosure are the same. In some such embodiments, a fusion polypeptide comprises two or more immunomodulatory polypeptides that at are different from one another.

[0082] Thus, in some embodiments, an immunomodulatory polypeptide may include more than one immune agonist moiety which, in various embodiments, may be the same or different. In some such Page 19 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 embodiments, two or more such immune agonist moieties are the same; in some embodiments, all such immune agonist moieties are the same. In some embodiments, an immunomodulatory polypeptide includes two or more immune agonist moieties that differ from one another; in some embodiments, no two such immune agonist moieties are the same.

[0083] In some embodiments, a fusion polypeptide comprises two or more immunomodulatory polypeptides (e.g., two or more immune agonist moieties) that include at least two subtypes of immunomodulatory polypeptides (e.g., immune agonist moieties) - for example so that the fusion polypeptide includes at least two of a first subtype and at least two of a second subtype. In some such embodiments, a subset of the same immunomodulatory polypeptides (e.g., immune agonist moieties) equals 1 immunomodulatory polypeptide out of 2 total moieties in the fusion polypeptide, 2 immunomodulatory polypeptides out of 2 total, 1 immunomodulatory polypeptides out of 3, 2 immunomodulatory polypeptides out of 3 total, or 3 immunomodulatory polypeptides out of 3. In some such embodiments, a subset of different immunomodulatory polypeptides equals 1 immune immunomodulatory polypeptides out of 2 total, 2 immunomodulatory polypeptides out of 2 total, 1 immunomodulatory polypeptides out of 3 total, 2 immunomodulatory polypeptides out of 3 total, or 3 immunomodulatory polypeptides out of 3 total.

[0084] In some embodiments, an immunomodulatory polypeptide (e.g., an immune agonist moiety) activates or inhibits activity of a cell of the immune system (e.g., is signaling competent). In some embodiments, an immunomodulatory polypeptide (e.g., an immune agonist moiety) is assessed, for example as part of a fusion polypeptide, e.g., as described herein.

[0085] For example, in some embodiments, signal competency is characterized in that, when assessed for binding to a particular binding partner, an immune agonist moiety or moieties or functional fragments thereof displays binding comparable or stronger than that of a reference standard (e.g., a wild-type polypeptide). For example, in some embodiments, signal competency is characterized in that, when assessed for a biological effect, e.g., in vitro or in vivo, an immune agonist moiety or moieties or functional fragments thereof displays said biological effect comparable to that of a reference standard (e.g., a wild-type polypeptide).

[0086] For example, in some embodiments, an immunomodulatory polypeptide (e.g., an immune agonist moiety) is an immune response stimulatory moiety. In some embodiments, a response stimulatory moiety is, for example, but without limitation, a ligand, a modified ligand, an agonistic antibody or an antibody agent, or a combination thereof.Page 20 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0087] In some embodiments, an immunomodulatory polypeptide is or comprises an antibody or antibody agent (e.g., antibody-format molecule). In embodiments, the antibody or antibody agent is any immunological binding moiety including, but not limited to, a scFv, Fab, Fab', Fab'2, Fab2, Fab3, F(ab’)2, Fd, Fd’, Fv, Feb, diabody, triabody, tetrabody, minibody, maxibody, tandab, DVD, BiTe, TandAb, single domain antibody (e.g., shark single domain antibody, such as IgNAR or fragments thereof), camelid antibody, full length antibody, masked antibody (e.g., Probodies®), Small Modular ImmunoPharmaceutical (SMIPTM), single chain or Tandem diabody (TandAb®), VHH, Anticalin®, Nanobody®, BiTE®, ankyrin repeat protein or DARPIN®, Avimer®, DART, TCR-like antibody, Adnectin®, Affilin®, Trans-body®, Affibody®, TrimerX®, MicroProtein, Fynomer®, Centyrin®, KALBITOR®, or any combination thereof. In embodiments, the antibody or antibodyformat molecule comprises Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv (scFv), diabody, single domain antibody (sdAb), single domain heavy chain antibody (VHH, VNAR), linear antibody, monoclonal antibody, polyclonal antibody, bispecific antibody, multi-specific antibody, chimeric antibody, humanized antibody, human antibody, non-human antibody, and fusion protein comprising an antigen-binding portion of an antibody.

[0088] In embodiments, agonists herein form a tertiary structure and / or quaternary structure that resembles an antibody (e.g., immunoglobulin). In embodiments, agonists herein fold into one or more compact, globular domains (e.g., one or more variable heavy domain and / or variable light domain) which are fused by one or more flexible hinge regions and / or amino acid linkers. In embodiments, agonists and herein form crosslinked structures via disulfide bond formation (e.g., resembling IgG antibody structures composed of four polypeptide chains: two heavy chains and two light chains, which form a substantially “Y” shaped tertiary / quaternary structure). In embodiments, the metal hydroxide-binding polypeptide is substantially a “random coil” domain that substantially lacks secondary structure, e.g., lacks a-helical content, p-sheet folds, etc., and the presence of the metal hydroxide-binding polypeptide does not interfere with antibody structure formation.

[0089] In some embodiments, an antibody agent comprises or is any polypeptide or peptide-containing molecule comprising at least a portion of an immunoglobulin including at least one complementarity determining region (CDR) of a VH or a VL derived from any antibody, e.g., an antibody described herein. In some embodiments, antigen-binding fragments comprise (i) a Fab fragment, a monovalent fragment comprising VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at a hinge region; (iii) a Fd fragment comprising VH and CH1 domains; (iv) a Fv fragment comprising VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragmentPage 21 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 comprising a VH domain; (vi) a camelid or camelized variable domain; (vii) a scFv, a fusion protein of VH and VL regions; (viii) a single domain antibody; or (viii) a heavy chain and a light chain (e.g., a half antibody)), or combinations thereof. In some embodiments, antigen-binding fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. In some embodiments, antibody or an antibody agent binds to at least one immune effector cell antigen described herein.

[0090] In some embodiments, an antibody or an antibody agent comprises a VH and a VL domain. In some embodiments, an antibody or antibody agent comprises an immunoglobulin molecule of four polypeptide chains, e.g., two heavy chains and two light chains. A heavy chain can include a VH domain and a heavy chain constant domain. A heavy chain constant domain can include CH1 , hinge, CH2, CH3, and optionally, a CH4 region. A light chain can include a VL domain and a light chain constant domain. A light chain constant domain can include a CL domain.

[0091] In some embodiments, a VH and / or a VL can be further subdivided into regions of variability, termed CDRs, interspersed with regions that are more conserved, termed framework regions (FR). Such VH and / or VL domains can each include three CDRs and four framework regions, arranged from amino-terminus to carboxyl-terminus in the following order: FR1 , CDR1, FR2, CDR2, FR3, CDR3, FR4, one or more of which can be engineered as described herein. In general, there are three CDRs in each VH region (HCDR1 , HCDR2, and HCDR3) and three CDRs in each VL region (LCDR1, LCDR2, and LCDR3). Framework region and CDRs can be defined using a number of well-known schemes (see, e.g., Kabat, E. A, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular’s AbM antibody modeling software, each of which is hereby incorporated by reference in its entirety). In embodiments, antibodies and antibody-format molecules herein comprise variation in a VH / VL or HC / LC while maintaining a set of 6 CDRs, e.g., having substitutions, deletions, or insertions in a framework or constant region. For example, in embodiments, antibodies herein may comprise about 90% to about 99% sequence identity to a VH / VL or HC / LC while having a defined set of 6 CDRs, where the sequence variations are in the sequences outside of the CDRs, e.g., mutations for humanization, half-life improvement, adapting the antigen-binding domain to a particular antibody-format molecule, etc.

[0092] In some embodiments, an antibody or an antibody agent can bind to at least one immune effector cell antigen described herein. Immune effector cells can include, but are not limited to, T cells (e.g., cytotoxicPage 22 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 or helper T cells), natural killer (NK) cells, macrophages, monocytes, dendritic cells (DCs), neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans' cells, or B-lymphocytes. In certain embodiments, at least one immune effector cell antigen comprises or is a T cell (e.g., cytotoxic or helper T cell) antigen. In certain embodiments, at least one immune effector cell antigen comprises or is a NK cell antigen. In certain embodiments, at least one immune effector cell antigen comprises or is a macrophage antigen. In certain embodiments, at least one immune effector cell antigen comprises or is a DC antigen. In certain embodiments, at least one immune effector cell antigen comprises or is a neutrophil antigen. In certain embodiments, at least one immune effector cell antigen comprises or is an eosinophil antigen. In certain embodiments, at least one immune effector cell antigen comprises or is a mast cell antigen. In certain embodiments, at least one immune effector cell antigen comprises or is a platelet antigen. In certain embodiments, at least one immune effector cell antigen comprises or is a large granular lymphocyte antigen. In certain embodiments, at least one immune effector cell antigen comprises or is a Langerhans' cell antigen. In certain embodiments, at least one immune effector cell antigen comprises or is a B-lymphocyte cell antigen.

[0093] In some embodiments, an antibody or an antibody agent binds specifically to a TNFRSF polypeptide. In some embodiments, TNFRSF polypeptide is or comprises, but is not limited to, TNFR1 , TNFR2, LTBR, 0X40, CD27, CD30, CD40, FasR, DCR1 , DCR2, DCR3, 4-1 BB, DR3, DR4, DR5, DR6, RANK, OPG, TWEAKR, TACI, BAFF-R, HVEM, p75NTR, BCMA, GITR, TNFRSF19 and TNFRSF27. In some embodiments, an antibody or an antibody agent binds specifically to TNFR1. In some embodiments, an antibody or an antibody agent binds specifically to TNFR2. In some embodiments, an antibody or an antibody agent binds specifically to LTBR. In some embodiments, an antibody or an antibody agent binds specifically to 0X40. In some embodiments, an antibody or an antibody agent binds specifically to CD27. In some embodiments, an antibody or an antibody agent binds specifically to CD30 In some embodiments, an antibody or an antibody agent binds specifically to CD40. In some embodiments, an antibody or an antibody agent binds specifically to FasR. In some embodiments, an antibody or an antibody agent binds specifically to DCR1. In some embodiments, an antibody or an antibody agent binds specifically to DCR2. In some embodiments, an antibody or an antibody agent binds specifically to DCR3. In some embodiments, an antibody or an antibody agent binds specifically to 4-1 BB. In some embodiments, an antibody or an antibody agent binds specifically to DR3. In some embodiments, an antibody or an antibody agent binds specifically to DR4. In some embodiments, an antibody or an antibody agent binds specifically to DR5. In some embodiments, an antibody or an antibody agent binds specifically to DR6. In some embodiments, an antibody or an antibody agent binds specifically to RANK. In some embodiments, an antibody or an antibody agent binds specifically to OPG. In some Page 23 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 embodiments, an antibody or an antibody agent binds specifically to TWEAKR. In some embodiments, an antibody or an antibody agent binds specifically to TACI. In some embodiments, an antibody or an antibody agent binds specifically to BAFF-R. In some embodiments, an antibody or an antibody agent binds specifically to HVEM. In some embodiments, an antibody or an antibody agent binds specifically to p75NTR. In some embodiments, an antibody or an antibody agent binds specifically to BCMA. In some embodiments, an antibody or an antibody agent binds specifically to GITR. In some embodiments, an antibody or an antibody agent binds specifically to TNFRSF19. In some embodiments, an antibody or an antibody agent binds specifically to TNFRSF27.

[0094] In embodiments, the antibody or antibody-format molecule and the metal hydroxide-binding polypeptide are fused. In embodiments, the metal hydroxide-binding polypeptide is fused to the C-terminus of a first chain of the antibody or antibody-format molecule. In embodiments, the first chain comprises a heavy chain or a heavy variable domain. Alternatively, in embodiments, this first chain comprises a light chain or a light variable domain.CD40 agonists

[0095] In some embodiments, an immunomodulatory polypeptide comprises a CD40 agonist (CD40mAb) immunomodulatory polypeptide (e.g., a CD40 immune agonist moiety). In some embodiments, a CD40 agonist is or comprises an antibody or an antibody agent. In some embodiments, a CD40 agonist is or comprises a CD40 ligand (CD40L).

[0096] In embodiments, described herein is an agonist comprising an antibody or antibody-format molecule that binds CD40 and metal hydroxide-binding polypeptide comprising an amino acid sequence having one or more phosphorylation sites. In embodiments, the agonist is an anti-CD40 fusion capable of agonism through a CD40 / CD40L cell signaling pathway.

[0097] Cluster of Differentiation 40 (CD40) is a costimulatory receptor that is expressed by antigen- presenting cells (APCs), B cells, and tumor cells, and it plays an important role in innate and adaptive immunity. CD40 interacts with CD40L, which is found primarily on activated CD4+ T cells. After its ligation, CD40 is activated and then binds to TNF receptor associated factors (TRAFs), which can propel a wide range of downstream signaling pathways with the subsequent production of surface and secreted molecules that ultimately impact on both humoral and cellular immunity, and eventually on inflammatory responses.Page 24 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0098] Engagement by CD40L expressed on T cells with CD40 expressed on DCs induces positive signaling that leads to expression of CD80 / 86 and the production of molecules that skews towards Th1 differentiation in CD4+ T cells. In addition to IL-12, CD40L signaling in T cells induces IFN-y production. DCs activated via the CD40 / CD40L axis also mediate crosstalk among multiple lymphocytes to regulate, for example, B cell class switching or regulatory T cell activation (May DY and Clark EA. Semin Immunol. 2010 Jun;22(3):190.).

[0099] CD40 plays important roles in the innate and adaptive immune response, and dysregulation of CD40 has been implicated in a number of disease states. Exemplary such disease states include, but are not limited to, autoimmune thyroiditis, type 1 diabetes, inflammatory bowel disease, psoriasis, multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus, cancer, traumatic brain injury, Alzheimer’s Disease, Parkinson’s Disease, stroke, epilepsy, nerve injury, multiple sclerosis, Amyotrophic lateral sclerosis and myasthenia gravis, (e.g., Peters et al. Semin Immunol. 2009. 21 (5) : 293-300; . and Ots et al. Int J Mol Sci. 2022. 23(8): 4115). Use of CD40 modulators as a therapeutic modality has been studied extensively, including for treatment of tumors (Li and Wang, Oncol. Lett. 20: 176, 2020; and Bullock, Cell and Mol Immunol. 2022. (19)14- 22. ). Currently, use of therapeutic CD40 agonists is hindered by the occurrence of adverse events, such as storms of cytokine release, hepatotoxicity and thromboembolic events, in patients treated with these antibodies. Furthermore, dose-dependent T-cell margination after CD40 agonist administration to patients has been observed, resulting in a counter-productive immune-dampening effects of the treatment. Intratumoral delivery of CD40 moieties lessens but does not abrogate the above adverse effects. Anchoring of CD40 agonist moieties with alum could improve safety and efficacy over conventional intratumorally-delivered CD40 antibodies.

[0100] In some embodiments, an immunomodulatory polypeptide disclosed herein is or comprises a CD40 immune agonist moiety. In some embodiments, an immunomodulatory polypeptide disclosed herein comprises a plurality of CD40 immune agonist moieties. In some embodiments, an immunomodulatory polypeptide disclosed herein is or comprises a CD40 ligand (CD40L). In some embodiments, an immunomodulatory polypeptide disclosed herein is or comprises a CD40 agonistic antibody or an antibody agent.

[0101] In some embodiments, an immunomodulatory polypeptide disclosed herein comprises an anti- CD40 antibody or an antibody agent. In some embodiments, an anti-CD40 antibody or an antibody agent binds to a CD40 receptor expressed by antigen-presenting cells. In some embodiments, antigen presenting cells thatPage 25 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 express CD40 are or comprise macrophages, B cells, T-cells, or dendritic cells. In some embodiments, an anti- CD40 antibody or an antibody agent binds to CD40 and / or competes for binding with a CD40 ligand (CD40L). In some embodiments, an anti-CD40 antibody or an antibody agent binds to CD40 and / or competes for binding with a CD40 ligand (CD40L) expressed by T-cells. In some embodiments, an anti-CD40 antibody or an antibody agent binds to CD40 and / or competes for binding with a CD40 ligand (CD40L) expressed by T-helper cells (CD4+ T-cells). In some embodiments, an anti-CD40 antibody or an antibody agent binds to a CD40 receptor expressed by cancer cells.

[0102] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 1 , 2, and / or 3. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 1 , 2, and / or 3. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 1 , 2, and / or 3.

[0103] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NO: 6, FTS, and / or SEQ ID NO: 8. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NO: 6, FTS, and / or SEQ ID NO: 8. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NO: 6, FTS, and / or SEQ ID NO: 8.

[0104] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having CDRs with amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, FTS, and / or SEQ ID NO: 8.

[0105] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 11 , 12, and / or 13. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 11, 12, and / or 13. In embodiments, the antibodyPage 26 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 11 , 12, and / or 13.

[0106] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 16, 17, and / or 18. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 16, 17, and / or 18. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 16, 17, and / or 18.

[0107] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having CDRs of SEQ ID NOs: 11 , 12, 13, 16, 17, and / or 18.

[0108] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 21 , 22, and / or 23. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 21, 22, and / or 23. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 21 , 22, and / or 23.

[0109] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 26, 27, and / or 28. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 26, 27, and / or 28. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 26, 27, and / or 28.

[0110] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having CDRs of SEQ ID NOs: 21 , 22, 23, 26, 27, and / or 28.

[0111] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected fromPage 27 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 31 , 32, and / or 33. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 31, 32, and / or 33. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 31 , 32, and / or 33.

[0112] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 36, 37, and / or 38. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 36, 37, and / or 38. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 36, 37, and / or 38.

[0113] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having CDRs of SEQ ID NOs: 31, 32, 33, 36, 37, and / or 38.

[0114] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 41 , 42, and / or 43. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 41, 42, and / or 43. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 41 , 42, and / or 43.

[0115] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 46, 47, and / or 48. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 46, 47, and / or 48. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 46, 47, and / or 48.

[0116] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having CDRs of SEQ ID NOs: 41 , 42, 43, 46, 47, and / or 48.Page 28 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0117] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NO: 51 , SEQ ID NO: 52, and / or LDY. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1, 2, or 3 CDRs having the amino acid sequence of SEQ ID NO: 51 , SEQ ID NO: 52, and / or LDY. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NO: 51 , SEQ ID NO: 52, and / or LDY.

[0118] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 56, 57, and / or 58. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 56, 57, and / or 58. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 56, 57, and / or 58.

[0119] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having CDRs of SEQ ID NO: 51 , SEQ ID NO: 52, LDY, SEQ ID NO: 56, SEQ ID NO: 57, and / or SEQ ID NO: 58.

[0120] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 61 , 62, and / or 63. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 61, 62, and / or 63. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 61 , 62, and / or 63.

[0121] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 66, 67, and / or 68. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 66, 67, and / or 68. In embodiments, the antibodyPage 29 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 66, 67, and / or 68.

[0122] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having CDRs of SEQ ID NOs: 61 , 62, 63, 66, 67, and / or 68.

[0123] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 71 , 72, and / or 73. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 71, 72, and / or 73. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 71 , 72, and / or 73.

[0124] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 76, 77, and / or 78. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 76, 77, and / or 78. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 76, 77, and / or 78.

[0125] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having CDRs of SEQ ID NOs: 71, 72, 73, 76, 77, and / or 78.

[0126] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 81 , 82, and / or 83. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 81, 82, and / or 83. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 81 , 82, and / or 83.

[0127] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected fromPage 30 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 86, 87, and / or 88. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 86, 87, and / or 88. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 86, 87, and / or 88.

[0128] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having CDRs of SEQ ID NOs: 81 , 82, 83, 86, 87, and / or 88.

[0129] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 91 , 92, and / or 93. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 91, 92, and / or 93. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 91 , 92, and / or 93.

[0130] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 96, 97, and / or 98. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 96, 97, and / or 98. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 96, 97, and / or 98.

[0131] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having CDRs of SEQ ID NOs: 91 , 92, 93, 96, 97, and / or 98.

[0132] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 101, 102, and / or 103. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 101 , 102, and / or 103. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 101 , 102, and / or 103.Page 31 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0133] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having an amino acid sequence with 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 106, 107, and / or 108. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 106, 107, and / or 108. In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having the CDRs of SEQ ID NOs: 106, 107, and / or 108.

[0134] In embodiments, the antibody or antibody-format molecule comprises an anti-CD40 binding domain having CDRs of SEQ ID NOs: 101 , 102, 103, 106, 107, and / or 108.

[0135] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH comprising one, two, or three HC CDR sequences each with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a HC CDR in Table 3; and / or (b) a VL comprising one, two, or three LC CDR sequences each with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a LC CDR in Table 3. In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising one, two, or three HC CDR sequences each with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a HC CDR of Table 3; and / or (b) a VL comprising one, two, or three LC CDR sequences each with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a LC CDR of Table 3. In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising one, two, or three HC CDR sequences each comprising or consisting of a HC CDR of Table 3; and / or (b) a VL comprising one, two, or three LC CDR sequences each comprising or consisting of a LC CDR of Table 3. In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a VH in Table 3; and / or (b) a VL with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a VL in Table 3. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a VH of Table 3; and / or (b) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a VL of Table 3. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH comprising or consisting of a VH of Table 3; and / or (b) a VL comprising or consisting of a VL of Table 3.Page 32 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0136] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a heavy chain with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a heavy chain in Table 3; and / or (b) a light chain with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a light chain in Table 3. In some embodiments, an antibody or antibody agent is or comprises: (a) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a heavy chain of Table 3; and / or (b) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a light chain of Table 3. In some embodiments, an antibody or antibody agent is or comprises: (a) a heavy chain comprising or consisting of a heavy chain of Table 3; and / or (b) a light chain comprising or consisting of a light chain of Table 3.

[0137] In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 1; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 2; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 3 and / or (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 6; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to FTS; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 8.

[0138] In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 11 ; a HC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 12; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 13 and / or (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 16; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 17; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 18.

[0139] In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 21 ; a HC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%,Page 33 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-500599%, or more identity to SEQ ID NO: 22; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 23 and / or (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 26; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 27; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 28.

[0140] In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 31 ; a HC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 32; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 33 and / or (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 36; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 37; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 38.

[0141] In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 41 ; a HC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 42; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 43 and / or (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 46; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 47; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 48.

[0142] In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 51 ; a HC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 52; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to LDY and / or (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 56; a LC CDR 2 with atPage 34 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 57; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 58.

[0143] In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 61 ; a HC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 62; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 63 and / or (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 66; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 67; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 68.

[0144] In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 71 ; a HC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 72; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 73 and / or (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 76; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 77; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 78.

[0145] In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 81 ; a HC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 82; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 83 and / or (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 86; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 87; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 88.Page 35 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0146] In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 91 ; a HC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 92; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 93 and / or (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 96; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 97; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 98.

[0147] In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 101; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 102; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 103 and / or (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 106; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 107; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 108.

[0148] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 4; and / or (b) a VL with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity SEQ ID NO: 9. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 4; and / or (b) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 9.

[0149] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 14; and / or (b) a VL with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity SEQ ID NO: 19. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQPage 36 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005ID NO: 14; and / or (b) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 19.

[0150] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 24; and / or (b) a VL with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity SEQ ID NO: 29. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 24; and / or (b) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 29.

[0151] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 34; and / or (b) a VL with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity SEQ ID NO: 39. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 34; and / or (b) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 39.

[0152] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 44; and / or (b) a VL with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity SEQ ID NO: 49. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 44; and / or (b) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 49.

[0153] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 54; and / or (b) a VL with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity SEQ ID NO: 59. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 54; and / or (b) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 59.Page 37 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0154] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 64; and / or (b) a VL with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity SEQ ID NO: 69. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 64; and / or (b) a VL with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 69.

[0155] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 74; and / or (b) a VL with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity SEQ ID NO: 79. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 74; and / or (b) a VL with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 79.

[0156] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 84; and / or (b) a VL with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity SEQ ID NO: 89. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 84; and / or (b) a VL with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 89.

[0157] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 94; and / or (b) a VL with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity SEQ ID NO: 99. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 94; and / or (b) a VL with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 99.

[0158] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a VH with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID Page 38 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005NO: 104; and / or (b) a VL with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity SEQ ID NO: 109. In some embodiments, an antibody or antibody agent is or comprises: (a) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 104; and / or (b) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 109.

[0159] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a heavy chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 5; and / or (b) a light chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 10. In some embodiments, an antibody or antibody agent is or comprises: (a) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 5; and / or (b) a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 10.

[0160] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a heavy chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 15; and / or (b) a light chain with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 20. In some embodiments, an antibody or antibody agent is or comprises: (a) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 15; and / or (b) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 20.

[0161] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a heavy chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 25; and / or (b) a light chain with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 30. In some embodiments, an antibody or antibody agent is or comprises: (a) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 25; and / or (b) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 30.

[0162] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a heavy chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 35; and / or (b) a light chain with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 40. In some embodiments, an antibody or antibody agent is Page 39 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 or comprises: (a) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 35; and / or (b) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 40.

[0163] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a heavy chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 45; and / or (b) a light chain with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 50. In some embodiments, an antibody or antibody agent is or comprises: (a) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 45; and / or (b) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 50.

[0164] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a heavy chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 55; and / or (b) a light chain with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 60. In some embodiments, an antibody or antibody agent is or comprises: (a) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 55; and / or (b) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 60.

[0165] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a heavy chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 65; and / or (b) a light chain with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 70. In some embodiments, an antibody or antibody agent is or comprises: (a) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 65; and / or (b) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 70.

[0166] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a heavy chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 75; and / or (b) a light chain with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 80. In some embodiments, an antibody or antibody agent is or comprises: (a) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,Page 40 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 or more identity to SEQ ID NO: 75; and / or (b) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 80.

[0167] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a heavy chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 85; and / or (b) a light chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 90. In some embodiments, an antibody or antibody agent is or comprises: (a) a heavy chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 85; and / or (b) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 90.

[0168] In some embodiments, a CD40 agonist antibody or antibody agent comprises: (a) a heavy chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 95; and / or (b) a light chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 100. In some embodiments, an antibody or antibody agent is or comprises: (a) a heavy chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 95; and / or (b) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 100.

[0169] In some embodiments, a CD40 agonist antibody or antibody agent comprises: a light chain with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 111 . In some embodiments, an antibody or antibody agent is or comprises: a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 111.

[0170] In some embodiments, a CD40 agonist antibody or antibody agent is or comprises an scFv. In some embodiments, an scFv has at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to sequences in Table 3. In some embodiments, an scFv has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to one or more sequences in Table 3.

[0171] In some embodiments, an scFv has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 112-121. In some embodiments, an scFv has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 112. In some embodiments, an scFv has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 113. In some embodiments, an scFv has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 114. In some embodiments, an Page 41 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 scFv has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 115. In some embodiments, an scFv has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 116. In some embodiments, an scFv has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 117. In some embodiments, an scFv has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 118. In some embodiments, an scFv has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 119. In some embodiments, an scFv has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 120. In some embodiments, an scFv has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 121.

[0172] In some embodiments, a CD40 agonist antibody or antibody agent is or comprises a diabody. In some embodiments, a diabody has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to sequences in Table 3. In some embodiments, a diabody has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to one or more sequences in Table 3.

[0173] In some embodiments, a diabody has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 122-127. In some embodiments, a diabody has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 122. In some embodiments, a diabody has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 123. In some embodiments, a diabody has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 124. In some embodiments, a diabody has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 125. In some embodiments, a diabody has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 126. In some embodiments, a diabody has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 127.

[0174] In some embodiments, a CD40 agonist moiety comprises a CD40 ligand (CD40L). In some embodiments, a CD40L has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to one or more sequences in Table 4. In some embodiments, a CD40L has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to one or more sequences in Table 4.Page 42 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0175] In some embodiments, a CD40 agonist moiety comprises a CD40L with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence selected from SEQ ID NOs: 220-228. In some embodiments, CD40 agonist moieties comprise a CD40L with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence selected from SEQ ID NOs: 220-228.

[0176] In some embodiments, a CD40L is a human CD40L. In some embodiments, a human CD40L has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence selected from SEQ ID NOs: 220-222. In some embodiments, a human CD40L has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence selected from SEQ ID NOs: 223-225.

[0177] In some embodiments, a human CD40L has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 220. In some embodiments, a human CD40L has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 221. In some embodiments, a human CD40L has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 222.

[0178] In some embodiments, a CD40L is a canine CD40L. In some embodiments, a canine CD40L has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence selected from SEQ ID NOs: 223-225. In some embodiments, a canine CD40L has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence selected from SEQ ID NOs: 223-225.

[0179] In some embodiments, a canine CD40L has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 223. In some embodiments, a canine CD40L has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 224. In some embodiments, a canine CD40L has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 225.

[0180] In some embodiments, a CD40L is a murine CD40L. In some embodiments, the murine CD40L has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence selected from SEQ ID NOs: 226-228. In some embodiments, a murine CD40L has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence selected from SEQ ID NOs: 226-228.Page 43 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0181] In some embodiments, a murine CD40L has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 226. In some embodiments, a murine CD40L has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 227. In some embodiments, a murine CD40L has at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 228.

[0182] In embodiments, the antibody or antibody-format molecule comprises one or more Fc domains.

[0183] In embodiments, the term “Fc,” “Fc domain,” and / or "Fc region" refers to the portion of a native immunoglobulin formed by the respective Fc domains (or Fc moieties) of its two heavy chains. In embodiments, these terms refer to a portion of a single immunoglobulin (Ig) heavy chain wherein the Fc domain does not comprise an Fv domain. In embodiments, these terms refer to a portion of a single immunoglobulin (Ig) heavy chain also comprising an Fv domain. As such, in embodiments, an Fc domain may be referred to as "Ig" or "IgG." In embodiments, an Fc domain begins in the hinge region just upstream of the papain cleavage site and ends at the C-terminus of the antibody. Accordingly, in embodiments, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In embodiments, an Fc domain comprises at least one of: a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In embodiments, an Fc domain comprises a complete Fc domain ( / .e., a hinge domain, a CH2 domain, and a CH3 domain). In embodiments, an Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In embodiments, an Fc domain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In embodiments, an Fc domain consists of a CH3 domain or portion thereof. In embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In embodiments, an Fc domain consists of a CH2 domain (or portion thereof) and a CH3 domain. In embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In embodiments, an Fc domain lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). In embodiments, an Fc domain herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy-chain. In embodiments, the Fc domain may be derived from an immunoglobulin of any species and / or any subtype, including, but not limited to, a human lgG1 , lgG2, lgG3, lgG4, IgD, IgA, IgE, or IgM antibody. In embodiments, human lgG1 constant region comprises the amino acid sequence of SEQ ID NOs: 190-191. In embodiments, the Fc domain of human lgG1 comprises the amino acid sequence of SEQ ID NO: 190, or a variant thereof. In embodiments, Fc domain encompasses native Fc and Fc variant molecules. In embodiments, with Fc variantsPage 44 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 and native Fc domains, the term Fc domain includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means. In embodiments, the assignment of amino acid residue numbers to an Fc domain (and / or other portions of antibodies or antibody-format molecules herein) is in accordance with the definitions of Kabat. See, e.g., Sequences of Proteins of Immunological Interest (Table of Contents, Introduction and Constant Region Sequences sections), 5th edition, Bethesda, MD:NIH vol.1 :647- 723 (1991); Kabat et al., "Introduction" Sequences of Proteins of Immunological Interest, US Dept of Health and Human Services, NIH, 5th edition, Bethesda, MD vol. I:xiii-xcvi (1991); Chothia & Lesk, J. Mol. Biol.196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989), each of which is herein incorporated by reference for all purposes.

[0184] In embodiments, the antibody or antibody-format molecule is or comprises an immunoglobulin class selected from IgG, IgA, IgE, IgD, IgY, or IgM. In embodiments, the IgG isotype is selected from lgG1 , lgG2, lgG3, and I gG4. In embodiments, the IgA isotype is selected from lgA1 or lgA2.

[0185] In embodiments, the antibody or antibody-format molecule comprises an lgG1 isotype. In embodiments, the antibody or antibody-format molecule comprises an lgG2 isotype.

[0186] In embodiments, the antibody or antibody-format molecule comprises one or more Fc domain with an amino acid sequence having about or at least 80%, about or at least 85%, about or at least 90%, about or at least 95%, about or at least 96%, about or at least 97%, about or at least 98%, or about or at least 99% sequence identity with one or more amino acid sequences in Table 4 (e.g., SEQ ID NOs: 190-213, which includes Fc domains, CH2-CH3, and hinges), or having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more changes relative to one or more amino acid sequences in Table 4 (e.g., SEQ ID NOs: 190-213). In embodiments, the antibody or antibody-format molecule comprises one or more Fc domain with an amino acid sequence selected from Table 4 (e.g., SEQ ID NOs: 190-213).

[0187] In embodiments, the antibody or antibody-format molecule comprises one or more mutations that alters, reduces, or ablates, binding to an Fc receptor (e.g., Fey receptors (FcyRs), neonatal Fc receptor (FcRn), etc.), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or antibody-dependent cellular phagocytosis (ADCP). In embodiments, the one or more mutations is located within a constant portion (e.g., heavy chain Fc domain) and / or within a framework region of a variable heavy domain or a variable light domain. In embodiments, agonists herein exhibits altered binding to an activating FcyR (e.g., Fcyl, Fcylla, or FcyRllla). In embodiments, the agonists herein exhibit altered binding affinity to an inhibitory FcyR (e.g., FcyRllb).Page 45 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005Exemplary amino acid substitutions which alter FcR, or complement binding activity, and that are useful herein, are disclosed for example PCT Publication WO 05 / 063815, which is incorporated by reference herein.

[0188] In embodiments, the agonists herein comprise an Fc variant having an amino acid substitution which alters the antigen-dependent effector functions of the polypeptide, in particular ADCC or complement activation, e.g., as compared to a wild-type Fc region. In embodiments, agonists herein exhibit decreased binding to FcR gamma when compared to cognate, wild-type polypeptides and, therefore, mediate reduced effector function. In embodiments, Fc variants with decreased FcRy binding affinity are expected to reduce effector function, and such molecules are also useful, for example, for treatment of conditions in which target cell destruction is undesirable, e.g., where normal cells may express target molecules, or where chronic administration of the agonist might result in unwanted immunological activation.

[0189] In embodiments, anti-CD40 agonists herein exhibit CD40 agonistic activity without enhancing platelet activation related toxicity. In embodiments, alum anchoring functions, in part, to dampen toxicities associated with effector functions of antibodies and / or antibody-format molecules. In embodiments, fusion to a metal hydroxide binding polypeptide and / or phosphorylation (agnostic of alum anchoring) functions, in part, to dampen toxicities associated with effector functions of antibodies and / or antibody-format molecules.

[0190] In embodiments, the antibody or antibody-format molecule comprises an Fc domain having one or more mutations at positions selected from 226, 229, 234, 235, 237, 238, 239, 248, 249, 252, 254, 255, 256,258, 265, 267, 268, 269, 270, 271, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 297, 298,301, 303, 305, 307, 312, 315, 322, 324, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 356, 360, 366,368, 373, 376, 378, 379, 382, 388, 389, 394, 398, 405, 407, 414, 416, 419, 430, 434, 435, 437, 438, and 439, or equivalent positions thereto (e.g., relative to the I gG1 Fc domain of SEQ ID NO: 190, the lgG2 Fc domain of SEQ ID NO: 192, or the lgG4 Fc domain of SEQ ID NO: 194).

[0191] In embodiments, an Fc domain includes changes in the region between amino acids 234-238, including the sequence LLGGP (SEQ ID NO: 363) at the beginning of the CH2 domain. In embodiments, the Fc variant alters Fc mediated effector function, such as dampening ADCC and / or decrease binding avidity for one or more Fc receptors. In embodiments, sequence changes closer to the CH2-CH3 junction, for example at positions such as K322 or P331, reduce or eliminate complement mediated cytotoxicity and / or alter avidity for FcR binding. In embodiments, an Fc domain incorporates changes at residues P238 and / or P331, e.g., substituting the wild-type prolines at these positions to serine. In embodiments, alterations in the hinge region at one or more of the three hinge cysteines, to encode CCC, SCC, SSC, SCS, or SSS at these residues can Page 46 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 also affect FcR binding and molecular homogeneity, e.g., by elimination of unpaired cysteines that contribution to destabilization of the folded protein.

[0192] In embodiments, the Fc region is mutated to alter binding, for example, as described in U.S. Patent No. 6,737,056, incorporated herein by reference in its entirety. In embodiments, changing Pro331 in I gG3 to Ser resulted in 6-fold lower affinity as compared to unmutated I gG3, indicating the involvement of Pro331 in FcyRI binding. In embodiments, amino acid modifications at positions 234, 235, 236, and 237, 297, 318, 320 and 322 are disclosed as potentially altering receptor binding affinity, for example, in U.S. Patent No. 5,624,821, incorporated herein by reference in its entirety. Further mutations are contemplated for use herein, e.g., such as those described in U.S. Patent Publication No. 2006 / 0235208, incorporated herein by reference in its entirety. In embodiments, additional mutations useful herein are described in U.S. Patent Publication No. 2006 / 0235208, incorporated herein by reference in its entirety. In embodiments, mutants at L234A and / or L235A are described, e.g., in U.S. Patent Publication No. 2003 / 0108548, incorporated herein by reference in its entirety. In embodiments, the described modifications herein are included either individually or in combination. In embodiments, the mutation comprises D265A in human lgG1.

[0193] In embodiments, the Fc domain is an lgG1 isotype and comprises a mutation of one or more residues selected from L234, L235, and P329, or equivalent positions thereto. In embodiments, the one or more mutations comprises one or more mutation selected from L234A, L235A, and P329G or P329A, optionally comprising L234A, L235A, and P329A (referred to as a “LALAPA” mutation), or L234A, L235A, and P329G (referred to as a “LALAPG” mutation). In embodiments, the Fc domain is an lgG1 isotype and comprises a mutation of one or more residues selected from M252, S254, and T256, or equivalent positions thereto. In embodiments, the one or more mutations comprises one or more mutation selected from M252Y, S254T, and T256E (e.g., “YTE” mutation).

[0194] In embodiments, the agonists herein comprise one or more mutations / variants that provide altered FcyRIIA and / or FcyRIIB binding. In embodiments, the FcyRIIA and / or FcyRIIB binding is increased. In embodiments, the antibody is I gG1 or lgG2 isotype. In embodiments, the antibody is an lgG1 isotype antibody having one or more mutations in an Fc domain at positions selected from G237, P238, S267, H268, P271, N297, L328, A330. In embodiments, the one or more mutations comprises one or more of G237D, P238D, S267E, H268D, P271G, N297A, L328F, A330R. In embodiments, the one or more mutations comprises a combination selection from N297A, S267E, S627E / L328F, G237D / P238D / P271 G / A330R, or G237D / P238D / H268D / P271 G / A330R. In embodiments, the antibody is an lgG2 isotype antibody having onePage 47 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 or more mutations in an Fc domain at positions selected from C127 and / or C232. In embodiments, the one or more mutations comprises C127S and / or C232S. In embodiments, one or more of these mutations to lgG1 or lgG2 enhances binding affinity specifically to hFcyRIIB, but not to hFcyRIIA. In embodiments, binding affinity anti-CD40 agonists and agonists comprises one or more measurements of Ka, Kd, or KD, for example, as measured by surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), isothermal titration calorimetry (ITC), and the like, for example, as explained in Dahan R, et al., ‘Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcyR Engagement,” Cancer Cell. (2016) Vol. 29, No. 6, pp. 820-31, the entire contents of which is incorporated by reference.

[0195] In embodiments, the one or more mutations to the Fc domain increase clearance (e.g., reduce half-life) of the anti-CD40 antibody or antibody-format molecule. In embodiments, the one or more mutations to the Fc domain decrease clearance (e.g., increase half-life) of the anti-CD40 antibody or antibody-format molecule. In embodiments, CD40 agonists herein exhibit improved pharmacokinetic profile compared to a cognate CD40 agonist that is anchored / complexed with a metal hydroxide.

[0196] In embodiments, the agonists herein comprise one or more amino acid substitution which alters the glycosylation profile of the protein. For example, in embodiments, the Fc domain comprises one or more mutations leading to reduced glycosylation (e.g., reduced N-linked and / or O-linked glycosylation) or comprises an altered glycoform relative to a cognate, wild-type Fc domain or portion (e.g., a low fucose or fucose-free glycan). In embodiments, the agonists herein comprise an amino acid substitution near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS, where X is any amino acid. Exemplary amino acid substitutions which reduce or alter glycosylation and that are useful herein are disclosed, for example, in Published PCT Application WO 05 / 018572 and Published US Application US 2007 / 0111281 , the contents of each of which are incorporated by reference herein. In embodiments, the agonist comprises at least one Fc domain having an engineered cysteine residue, or analog thereof, which is located at a solvent-exposed boundary / surface. In embodiments, the agonists herein comprise an Fc domain having at least one engineered free cysteine residue, or analog thereof, that is substantially free of disulfide bonding with a second cysteine residue (e.g., unpaired). In embodiments, engineered cysteine residues, or analogs thereof, are subsequently conjugated to a functional domain using art-recognized techniques (e.g., conjugated with a thiol-reactive heterobifunctional stabilizing domain, functionalization through maleimide chemistry, iodoacetamides, etc.).Page 48 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0197] In embodiments, agonists herein comprise a fused Fc domain having two or more of its constituent Fc domains independently selected from the Fc domains described herein. In embodiments, the Fc domains are the same. In embodiments, the Fc domains are different. For example, in embodiments, the Fc domains comprise the same number of amino acid residues or they may differ in length by one or more amino acid residues (e.g., by about or at least about 1 , 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 residues). In embodiments, the Fc domains differ in sequence at one or more amino acid positions, e.g., “knob-in-hole” (KIH) antibody mutations, such as at positions C226, C229, T366, L368, T394, F405, and / or Y407. In embodiments, the Fc domains differ at about or at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or about 50 positions.CD40 agonist fusion polypeptides

[0198] In some embodiments, fusion polypeptides described herein comprise a TNFRSF agonist polypeptide and an alum-binding peptide. In some embodiments, a fusion polypeptide comprises a CD40 agonist moiety. In some embodiments, a fusion polypeptide comprises an antibody, an antibody agent, and / or a ligand. In embodiments, CD40 agonist fusions comprise any two or more polypeptides herein, e.g., an anti- CD40 binding domain and Fc domain, an anti-CD40 heavy chain or light chain and metal hydroxide-binding polypeptide, etc.

[0199] In some embodiments, a fusion polypeptide comprises an ABP with at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to one or more of SEQ ID NOs: 128-143. In some embodiments, a fusion polypeptide comprises a CD40 agonist antibody or antibody agent. In some embodiments, a fusion polypeptide comprise (a) a VH comprising one, two, or three HC CDR sequences each with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a HC CDR in Table 3; and (b) a VL comprising one, two, or three LC CDR sequences each with at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a LC CDR in Table 3. In some embodiments, a fusion polypeptide comprises (a) a VH comprising one, two, or three HC CDR sequences each with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a HC CDR in Table 3; and (b) a VL comprising one, two, or three LC CDR sequences each with at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a LC CDR in Table 3.

[0200] In some embodiments, a fusion polypeptide comprises (a) a VH comprising one, two, or three HC CDR sequences each with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a HC CDR of Table 3; and (b) a VL comprising one, two, or three LC CDR sequences each with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a LC CDR of Table 3.Page 49 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0201] In some embodiments, a fusion polypeptide comprises (a) a VH comprising one, two, or three HC CDR sequences each comprising or consisting of a HC CDR of T able 3; and (b) a VL comprising one, two, or three LC CDR sequences each comprising or consisting of a LC CDR of Table 3.

[0202] In some embodiments, a fusion polypeptide comprises: I. (a) a VH comprising: i) HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 1; ii) HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 2; and iii) HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 3; and (b) a VL comprising: i) LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 6; ii) LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to FTS; and iii) LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 8; II. (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 11 ; a HC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 12; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 13 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ I D NO: 16; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 17; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 18; III. (a) a VH comprising a HC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 21; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 22; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 23 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 26; a LC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 27; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 28; IV. In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 31; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 32; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 33 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, Page 50 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-500597%, 98%, 99%, or more identity to SEQ ID NO: 36; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 37; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 38; V. In some embodiments, an antibody or antibody agent described herein is or comprises: (a) a VH comprising a HC CDR1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 41 ; a HC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 42; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 43 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 46; a LC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 47; a LC CDR 3 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 48; VI. (a) a VH comprising a HC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 51; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 52; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to LDY and (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 56; a LC CDR2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 57; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 58; VII. (a) a VH comprising a HC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 61; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 62; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 63 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 66; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 67; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 68; VIII. (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 71; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 72; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 73 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 76; a LC CDR 2 with at least about 90%, 91%, 92%,Page 51 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-500593%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 77; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 78. IX. (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 81 ; a HC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 82; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 83 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 86; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 87; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 88; X. (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 91; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 92; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 93 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 96; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 97; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 98; or XI. (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 101; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 102; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 103 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 106; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 107; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 108.

[0203] In some embodiments, a fusion polypeptide comprises: (a) a VH comprising: i) HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 1; ii) HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 2; and ill) HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identityto SEQ ID NO: 3; and (b) a VL comprising: i) LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 6; ii) LC CDR 2 with at least about 90%,Page 52 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-500591%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to FTS; and iii) LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 8.

[0204] In some embodiments, a fusion polypeptide comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 11; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 12; a HC CDR 3 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 13 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 16; a LC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 17; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 18.

[0205] In some embodiments, a fusion polypeptide comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 21; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 22; a HC CDR 3 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 23 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 26; a LC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 27; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 28.

[0206] In some embodiments, a fusion polypeptide comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 31; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 32; a HC CDR 3 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 33 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 36; a LC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 37; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 38.

[0207] In some embodiments, a fusion polypeptide comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 41; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 42; a HC CDR 3 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more Page 53 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 identity to SEQ ID NO: 43 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 46; a LC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 47; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 48

[0208] In some embodiments, a fusion polypeptide comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 51; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 52; a HC CDR 3 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to LDY and (b) a VL comprising a LC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 56; a LC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 57; a LC CDR 3 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 58.

[0209] In some embodiments, a fusion polypeptide comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 61; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 62; a HC CDR 3 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 63 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 66; a LC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 67; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 68.

[0210] In some embodiments, a fusion polypeptide comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 71; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 72; a HC CDR 3 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 73 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 76; a LC CDR 2 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 77; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 78.

[0211] In some embodiments, a fusion polypeptide comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 81; a HC Page 54 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 82; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 83 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 86; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 87; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 88.

[0212] In some embodiments, a fusion polypeptide comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 91; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 92; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 93 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 96; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 97; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 98.

[0213] In some embodiments, a fusion polypeptide comprises: (a) a VH comprising a HC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 101; a HC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 102; a HC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 103 and (b) a VL comprising a LC CDR 1 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 106; a LC CDR 2 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 107; a LC CDR 3 with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 108.

[0214] In some embodiments, a fusion polypeptide comprises: I. (i) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 4; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 9; II. (i) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 14; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 19; III. (i) a VH with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 24; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,Page 55 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-500598%, 99%, or more identity to SEQ ID NO: 29; IV. (i) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 34; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 39; V. (i) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 44; and (ii) a VL with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 49; VI. (i) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 54; and (ii) a VL with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 59; VII. (i) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 64; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 69; VIII. (i) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 74; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 79; IX. (i) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 84; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 89; X. (i) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 94; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more identity to SEQ ID NO: 99; or XI. (i) a VH with at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, or 98%, 99%, or more identity to SEQ ID NO: 104; and (ii) a VL with at least about 90%, 91 %,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 109.

[0215] In some embodiments, a fusion polypeptide comprises: (i) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 4; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 9.

[0216] In some embodiments, a fusion polypeptide comprises: (i) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 14; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 19.

[0217] In some embodiments, a fusion polypeptide comprises: (i) a VH with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 24; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 29.Page 56 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0218] In some embodiments, a fusion polypeptide comprises: (i) a VH with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 34; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 39.

[0219] In some embodiments, a fusion polypeptide comprises: (i) a VH with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 44; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 49.

[0220] In some embodiments, a fusion polypeptide comprises: (i) a VH with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 54; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 59.

[0221] In some embodiments, a fusion polypeptide comprises: (i) a VH with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 64; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 69.

[0222] In some embodiments, a fusion polypeptide comprises: (i) a VH with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 74; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 79.

[0223] In some embodiments, a fusion polypeptide comprises: (i) a VH with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 84; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 89.

[0224] In some embodiments, a fusion polypeptide comprises: (i) a VH with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 94; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 99.

[0225] In some embodiments, a fusion polypeptide comprises: (i) a VH with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, 99%, or more identity to SEQ ID NO: 104; and (ii) a VL with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 109.

[0226] In some embodiments, a fusion polypeptide comprises: I. (i) a heavy chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 5; and (ii) a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 10; II. (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 15; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, Page 57 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-500597%, 98%, 99% or more identity to SEQ ID NO: 20; III. (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 25; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 30; IV. (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 35; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 40; V. (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 45; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 50; VI. (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 55; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 60; VII. (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 65; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 70; VIII. (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 75; and (ii) a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 80; IX. (i) a heavy chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 85; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 90; X. (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 95; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 100; or XI. a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 111.

[0227] In some embodiments, a fusion polypeptide comprises: (i) a heavy chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ I D NO: 5; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 10.

[0228] In some embodiments, a fusion polypeptide comprises: (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 15; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 20.Page 58 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0229] In some embodiments, a fusion polypeptide comprises: (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 25; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 30.

[0230] In some embodiments, a fusion polypeptide comprises: (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 35; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 40.

[0231] In some embodiments, a fusion polypeptide comprises: (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 45; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 50.

[0232] In some embodiments, a fusion polypeptide comprises: (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 55; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 60.

[0233] In some embodiments, a fusion polypeptide comprises: (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 65; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 70.

[0234] In some embodiments, a fusion polypeptide comprises: (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 75; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 80.

[0235] In some embodiments, a fusion polypeptide comprises: (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 85; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 90.Page 59 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0236] In some embodiments, a fusion polypeptide comprises: (i) a heavy chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 95; and (ii) a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 100.

[0237] In some embodiments, a fusion polypeptide comprises a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 111.

[0238] In some embodiments, a fusion polypeptide comprises: I. a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 147-148 and a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 10; II. a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 153-154 and a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 20; III. a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 157-158 and a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 30; IV. a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 161-162 and a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 40; V. a polypeptide with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 146-147 and a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 50; VI. a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 165-166 and a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 60; VII. a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 169-170 and a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 70; VIII. a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 173-174 and a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 80; IX. a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 177-178 and a light chain with at least about 90%, 91%,Page 60 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-500592%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 90; or X. a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 181-182 and a light chain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 100.

[0239] In some embodiments, a fusion polypeptide comprises: a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 147-148 and a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 10.

[0240] In some embodiments, a fusion polypeptide comprises: a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 153-154 and a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 20.

[0241] In some embodiments, a fusion polypeptide comprises: a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 157-158 and a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 30.

[0242] In some embodiments, a fusion polypeptide comprises: a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 161-162 and a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 40.

[0243] In some embodiments, a fusion polypeptide comprises: a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 146-147 and a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 50.

[0244] In some embodiments, a fusion polypeptide comprises: a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 165-166 and a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 60.Page 61 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0245] In some embodiments, a fusion polypeptide comprises: a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 169-170 and a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 70.

[0246] In some embodiments, a fusion polypeptide comprises: a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 173-174 and a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 80.

[0247] In some embodiments, a fusion polypeptide comprises: a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 177-178 and a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 90.

[0248] In some embodiments, a fusion polypeptide comprises: a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 181-182 and a light chain with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 100.

[0249] In some embodiments, fusion polypeptides described herein comprise an antibody or an antibody agent. In some embodiments, the antibody agent comprises a scFv or a diabody.

[0250] In some embodiments, a fusion polypeptide comprises a scFv. In some embodiments, a scFv has a sequence with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to scFv sequences from Table 3. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from: I. SEQ ID NO: 146; II. SEQ ID NO: 152; II. SEQ ID NO: 156; IV. SEQ ID NO: 160; V. SEQ ID NO: 164; VI. SEQ ID NO: 168; VII. SEQ ID NO: 172; VIII. SEQ ID NO: 176; IX. SEQ ID NO: 180; or X. SEQ ID NO: 184.

[0251] In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 146. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 152. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity toPage 62 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005SEQ ID NO: 156. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 160. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 164. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 169. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 172. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 176. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 180. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 184.

[0252] In some embodiments, a fusion polypeptide comprises a diabody. In some embodiments, a diabody has a sequence with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to diabody sequences from Table 3. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to sequences selected from: I. SEQ ID NO: 149; II. SEQ ID NO: 150; III. SEQ ID NO: 188; IV. SEQ ID NO: 189. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 149. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 150. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 188. In some embodiments, a fusion polypeptide comprises a polypeptide with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 189.

[0253] In some embodiments, a fusion polypeptide comprises an antibody agent with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 110.

[0254] In some embodiments, fusion polypeptides described herein comprise a CD40 ligand (CD40L) moiety. In some embodiments, a CD40L moiety is a native ligand. In some embodiments, a CD40L moiety is a modified ligand. In some embodiments, a CD40L moiety comprises a sequence with at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NOs: 220-228.Page 63 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0255] In some embodiments, fusion polypeptides described herein comprise one or more CD40L moieties. In some embodiments, fusion polypeptides described herein comprise one CD40L moiety. In some embodiments, fusion polypeptides described herein comprise one or more CD40L moieties. In some embodiments, fusion polypeptides described herein comprise two or more CD40L moieties. In some embodiments, fusion polypeptides described herein comprise three or more CD40L moieties. In some embodiments, two or more CD40L moieties are linked. In some embodiments, three or more CD40L moieties are linked. In some embodiments, CD40L moieties are linked with a linker.

[0256] In some embodiments, a fusion polypeptide comprises a CD40L moiety and an ABP. In some embodiments, a fusion polypeptide has at least at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence selected from SEQ ID NOs: 229-240.

[0257] In some embodiments, it is useful to generate fusion polypeptides comprising immunoglobulin G (lgG1) Fc moieties. In some embodiment, a fusion polypeptide described herein comprises an lgG1 Fc, lgG2 Fc, or IgG Fc moiety.

[0258] In some embodiment, a fusion polypeptide described herein comprises an lgG1 Fc moiety. In some embodiments, an lgG1 Fc moiety is a human, a canine or a murine lgG1 Fc moiety. In some embodiments, an lgG1 Fc moiety has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs 190-191. In some embodiments, an lgG1 Fc moiety has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs 190-191 , 196-197, or 202-203. In some embodiments, an lgG1 Fc moiety has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs 202-203.

[0259] In some embodiment, a fusion polypeptide described herein comprises an lgG2 Fc moiety. In some embodiments, the lgG2 Fc moiety is a human, a canine or a murine lgG2 Fc moiety. In some embodiments, the lgG2 Fc moiety has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 192-193. In some embodiments, the lgG2 Fc moiety has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 198-199. In some embodiments, the lgG2 Fc moiety has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 204-205.

[0260] In some embodiment, a fusion polypeptide described herein comprises an lgG4 Fc moiety. In some embodiments, the lgG4 Fc moiety is a human or a canine I gG4 Fc moiety. In some embodiments, the Page 64 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005I gG4 Fc moiety has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 194-195. In some embodiments, the lgG4 Fc moiety has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 200-201. or more

[0261] In some embodiments, the IgG Fc moiety comprises a hinge region. In some embodiments, the hinge region is defined as a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NOs: 206-213. In some embodiments, the IgG Fc moiety does not comprise a hinge region.

[0262] In some embodiments, a fusion polypeptide comprises one or more CD40L moieties and an IgG Fc moiety. In some embodiments, a fusion polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 214-284.

[0263] In some embodiments, a fusion polypeptide comprises one or more CD40L moieties and an lgG1 Fc moiety. In some embodiments, a fusion polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 241-260.

[0264] In some embodiments, a fusion polypeptide comprises one or more CD40L moieties and an lgG2 Fc moiety. In some embodiments, a fusion polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 261-272.

[0265] In some embodiments, a fusion polypeptide comprises one or more CD40L moieties and an lgG4 Fc moiety. In some embodiments, a fusion polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 273-284.

[0266] In some embodiments, it may be useful to generate fusion polypeptides comprising one or more T4 fibritin foldon domain (T4D) moieties. In some embodiments, a T4D moiety comprises a modified T4D sequence with at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 218. In some embodiments, a fusion polypeptide comprises one or more CD40L moieties linked at the 3’ end to a T4D moiety. In some embodiments, a fusion polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to sequences selected from SEQ ID NOs: 285- 300.

[0267] In some embodiments, it may be further useful to generate fusion polypeptides comprising one or more CD40L moieties, a T4D moiety and an IgG Fc moiety. In some embodiments, a fusion polypeptide comprises at least one CD40L linked at the 3’ end to a T4D moiety, wherein the T4D moiety is further linkedPage 65 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 at the 3’ end to a lgG1 Fc moiety. In some embodiments, a fusion polypeptide has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to sequences selected from SEQ ID NOs: 301-304.

[0268] In some embodiments, it may be useful to generate fusion polypeptides comprising one or more modified GCN4-derived leucine zipper (GCN4) moieties. In some embodiments, a T4D moiety comprises a modified GCN4 sequence with at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 219. In some embodiments, a fusion polypeptide comprises one or more CD40L moieties linked at the 3’ end to a GCN4 moiety. In some embodiments, a fusion polypeptide has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to sequences selected from SEQ ID NOs: 205-320.

[0269] In some embodiments, it may be further useful to generate fusion polypeptides comprising one or more CD40L moieties, a GCN4 moiety and an IgG Fc moiety. In some embodiments, a fusion polypeptide comprises one or more CD40L moieties linked at the 3’ end to a GCN4 moiety, wherein the GCN4 moiety is further linked at the 3’ end to a lgG1 Fc moiety. In some embodiments, a fusion polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to sequences selected from SEQ ID NOs: 321-324.

[0270] In some embodiments, a fusion polypeptide described herein comprises an ABP with SEQ ID NO: 141. In some embodiments, a fusion polypeptide described herein comprises an ABP with SEQ ID NO: 143.

[0271] In some embodiments, a fusion polypeptide described herein comprises a histidine tag.Metal-hydroxide binding polypeptide

[0272] In embodiments, the metal hydroxide-binding polypeptide comprises about or at least about 3 amino acids to about or at least about 100 amino acids. In embodiments, this length includes the one or more phosphorylation sites and / or spacer sequences therebetween and / or adjacent thereto. In embodiments, the metal hydroxide-binding polypeptide comprises about or at least about 3 amino acids, about or at least about 5 amino acids, about or at least about 10 amino acids, about or at least about 15 amino acids, about or at least about 20 amino acids, about or at least about 25 amino acids, about or at least about 30 amino acids, about or at least about 35 amino acids, about or at least about 40 amino acids, about or at least about 45 amino acids, about or at least about 50 amino acids, about or at least about 55 amino acids, about or at least about 60 amino acids, about or at least about 65 amino acids, about or at least about 70 amino acids, about or atPage 66 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 least about 75 amino acids, about or at least about 80 amino acids, about or at least about 85 amino acids, about or at least about 90 amino acids, about or at least about 95 amino acids, or about or at least about 100 amino acids, or any length therebetween.

[0273] In embodiments, the metal hydroxide-binding polypeptide comprises about or at least about 1, about or at least about 2, about or at least about 3, about or at least about 4, about or at least about 5, about or at least about 6, about or at least about 7, about or at least about 8, about or at least about 9, about or at least about 10, about or at least about 11 , about or at least about 12, about or at least about 13, 1 about or at least about 4, or about or at least about 15 or more phosphorylation sites (e.g., kinase target motifs). In embodiments, the metal hydroxide-binding polypeptide comprises about or at least about 2, about or at least about 4, about or at least about 6, or about or at least about 8 phosphorylation sites (e.g., kinase target motifs), optionally comprising less than about 10 phosphorylation sites (e.g., kinase target motifs). In embodiments, the metal hydroxide-binding polypeptide comprises about or at least about 1 , about or at least about 2, about or at least about 3, about or at least about 4, about or at least about 5, about or at least about 6, about or at least about 7, about or at least about 8, about or at least about 9, about or at least about 10, about or at least about 11 , about or at least about 12, about or at least about 13, 1 about or at least about 4, or about or at least about 15 or more phosphorylated residues (e.g., phosphorylated kinase target motifs).

[0274] In embodiments, the metal hydroxide-binding polypeptide comprises at least 1 phosphorylated amino acid, at least 2 phosphorylated amino acids, at least 3 phosphorylated amino acids, at least 4 phosphorylated amino acids, at least 5 phosphorylated amino acids, at least 6 phosphorylated amino acids, at least 7 phosphorylated amino acids, at least 8 phosphorylated amino acids, at least 9 phosphorylated amino acids, at least 10 phosphorylated amino acids, at least 11 phosphorylated amino acids, at least 12 phosphorylated amino acids, at least 13 phosphorylated amino acids, at least 14 phosphorylated amino acids, or at least 15 phosphorylated amino acids.

[0275] In embodiments, the one or more phosphorylation sites comprises one or more of a serine, threonine, or tyrosine that is configured to be phosphorylated (phosphoserine, phosphothreonine, or phosphotyrosine). In embodiments, one or more of the phosphorylated amino acids is selected from phosphoserine, phoshotyrosine, and phosphothreonine. In embodiments, the phosphorylated amino acid is phosphoserine.

[0276] In embodiments, the one or more phosphorylation sites comprises one or more kinase target motifs, optionally wherein the one or more kinase target motifs that is phosphorylated by a kinase selected Page 67 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 from a group consisting of: Fam20C, protein kinase A (PKA), cAMP-dependent protein kinase, cyclin- dependent kinase, extracellular-regulated kinase-2 (ERK2), casein kinase 1 (CK1), casein kinase 2 (CK2), glycogen synthase kinase-3, calmodulin-dependent protein kinase-2 (CaMK2), Abelson murine leukemia virus tyrosine kinase (ABL), epidermal growth factor receptor (EGFR), rous sarcoma virus tyrosine kinase (Src), insulin receptor tyrosine kinase (IPK), protein kinase B (PKB / AKT), protein kinase D (PKD), proviral integration site kinase 1-3 (PIM1-3), AMP-activated protein kinase, mitogen-activated protein kinase, and NimA-related kinase.

[0277] In embodiments, the one or more kinase target motifs comprises an amino acid consensus sequence selected from R-R-X-S / T-cp (protein kinase A (PKA)), R-R / K-X-S-cp (cAMP-dependent protein kinase), S / T-P-X-K / R (cyclin-dependent kinase (CDK), P-X-S / T-P (extracellular-regualted kinase-2 (ERK)), pS- X-X-S / T (casein kinase 1 (CK1)), D / E-D / E-D / E-X-X-S / T-cp-pS / pT-X-X-S / T-cp (CK1), S / T-D / E-X-D / E (casein kinase 2 (CK2)), S-X-X-X-pS (glycogen synthase kinase-3 (GSK3)), R-X-X-S / T (calmodulin-dependent protein kinase-2 (CaMK2), l / V / L-Y-X-X-P / F (Abelson murine leukemia virus tyrosine kinase (ABL), E-E-E-Y-F (epidermal growth factor receptor (EGFR)), E-E-l-Y-E / G-X-F (rous sarcoma virus tyrosine kinase (Src)), Y-M- M-M (insulin receptor tyrosine kinase (IPK)), R-X-R-X-X-S / J (protein kinase B (PKB / AKT)), L / l-X-R-X-X-S / J (protein kinase D (PKD)), R-X- R-X-X-S / T (proviral integration site kinases 1-2 (PIM1-3)), <p-X-R_X-X-S-X-X- X-l / L (AMP-activated protein kinase (AMPK), P / cp-X-S / T-P (mitogen-activated protein kinase), and (p-X-X-S / T (NimA-related kinase), wherein X is any amino acid, F is a hydrophobic amino acid, pS is a phosphoserine, pT is a phosphothreonine, and a phosphoacceptor amino acid is underlined.

[0278] In embodiments, the one or more phosphorylation sites (e.g., also referred to as “kinase target motif”) has a sequence that conforms to one or more consensus sequence selected from Table 1.Table 1 : Illustrative kinase target motifs of cellular kinases useful in metal hydroxide binding polypeptides herein. The phosphoacceptor amino acid(s) is underlined; (p = hydrophobic amino acid; X = any amino acid; pS or pT = priming phosphoserine or phosphothreonine, respectively.Page 68 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0279] In embodiments, the one or more phosphorylation sites comprises one or more kinase target motifs that is phosphorylated by a human secretory pathway kinases, e.g., including but not limited to, four- jointed box kinase 1 , Fam20A, Fam20B, Fam20C (also referred to as Golgi casein kinase), Fam198A, Fam198B, Fam69A, Fam69B, Fam69C, and vertebrate lonesome kinase (VLK). In embodiments, agonists herein comprise a metal hydroxide-binding peptide comprising two or more kinase target motifs of the secretory pathway kinase Fam20C. In embodiments, Fam20C is a human kinase that has been identified for phosphorylation of more than 100 secreted phosphoproteins, which comprises a substantial portion of the extracellular phosphoproteome. In embodiments, Fam20C phosphorylates casein and is often referred to as Golgi enriched fraction casein kinase. In embodiments, agonists herein are engineered to contain two or more phosphorylation sites that are phosphorylated by a Fam20C kinase, including for example, kinase target motifs comprising the amino acid sequence Ser-X-Glu (e.g., S-X-E), Ser-X-pSer (e.g., S-X-pS), and Ser-X-GIn-X-X- Asp-Glu-Glu (S-X-Q-X-X-D-E-E, SEQ ID NO: 364), where X is any amino acid, and pS is phosphorylated serine. In embodiments, agonists herein comprises a peptide substrate derived from casein (e.g., -casein) comprising one or more repeats of the amino acid sequence KKIEKFQSEEQQQ (SEQ ID NO: 369), or smaller portions thereof comprising an S-X-E kinase target motif, wherein the serine is phosphorylated by Fam20C.

[0280] In embodiments, the one or more phosphorylation sites comprises one or more kinase target motifs that is phosphorylated by a Fam20C group kinase. In embodiments, the metal hydroxide-binding polypeptide comprises an amino acid sequence having one or more phosphorylation sites comprising an aminoPage 69 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 acid consensus sequence of S-X-E, S-E-pS, or S-X-Q-X-X-D-E (SEQ ID NO: 365), wherein X is any amino acid and pS is a phosphoserine. In embodiments, X is selected from E, S, V, H, Q, and G, optionally wherein X is E. In embodiments, the one or more phosphorylation sites comprises S-E-E.

[0281] In embodiments, the metal hydroxide-binding peptide comprises at least one kinase target motif comprising an amino acid sequence of X1-X2-S-X3-E-X4-X5, wherein Xi is F, M or G; X2 is Q, E or G; X3 is E, S, V, H, Q and G; X4 is Q, S or G; and X5 is Q, N, or G, and wherein at least one serine is modified with a phosphate. In embodiments, X3 is E. In embodiments, Xi is F; and X2 is Q. In embodiments, Xi is M; and X2 isE. In embodiments, Xi is G; and X2 is G. In embodiments, X4 is Q; X5 is Q. In embodiments, X4 is E; X5 is S. In embodiments, X4 is G; X5 is G.

[0282] In embodiments, the metal hydroxide-binding peptide comprises at least one kinase target motif comprising an amino acid sequence of X1-X2-S-X3-E-X4-X5 IJ-S-X3-E- X6-X7 (SEQ ID NO: 370), wherein Xi isF, M or G; X2 is Q, E or G; X3 is E, S, V, H, Q and G; X4 is Q, S or G; X5 is Q, N, or G; X5 is G and Xe is G, and wherein L is a leucine or a peptide linker, as described herein.

[0283] In embodiments, the metal hydroxide-binding polypeptide is or comprises one or more amino acid sequence of XXSXEXX or XXSEEXX, wherein X is any amino acid. In embodiments, the metal hydroxide- binding polypeptide is or comprises one or more amino acid sequence of GGSEEGG (SEQ ID NO: 371), FQSEEQQ (SEQ ID NO: 372), or MESEESN (SEQ ID NO: 373). In embodiments, the metal hydroxide-binding polypeptide comprises two or more repeats of GGSEEGG (SEQ ID NO: 371), FQSEEQQ (SEQ ID NO: 372), or MESEESN (SEQ ID NO: 373). In embodiments, the metal hydroxide-binding polypeptide comprises one or more repeats of GGSEEGG (SEQ ID NO: 371).

[0284] In embodiments, the metal hydroxide-binding polypeptide comprises an amino acid sequence having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 128-143, 371 - 396, and 399-401, or a sequence having 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes thereto. In embodiments, the metal hydroxide-binding polypeptide is or comprises an amino acid sequence of any one of SEQ ID NOs: 128-143, 371-396, and 399-401.

[0285] In embodiments, the metal hydroxide-binding polypeptide comprises an amino acid sequence selected from Table 2.Page 70 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005Table 2: Illustrative metal hydroxide binding polypeptide sequences and motifs.Page 71 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0286] In embodiments, the metal hydroxide-binding polypeptide comprises 2 repeats, 3 repeats, 4 repeats, 5 repeats, 6 repeats, 7 repeats, 8 repeats, 9 repeats, 10 repeats, 11 repeats, 12 repeats, 13 repeats, 14 repeats, or 15 or more repeats. In embodiments, the repeat is of one or more phosphorylation sites described herein, or one or more of the sequences of Table 2.

[0287] In embodiments, the metal hydroxide-binding polypeptide is or comprises a sequence from the published PCT Application, WO2020263399, the entire contents are incorporated herein by reference, which shows that hydroxyl replacement (e.g., with phosphate groups) can increase a polypeptide’s adsorption via ligand exchange with a metal hydroxide (e.g., aluminum hydroxide), and furthermore can improve tumor retention and anti-tumor efficacy of such polypeptide (e.g., specifically of a fusion polypeptide comprising an immunomodulatory polypeptide and a metal-hydroxide-binding polypeptide in which such hydroxyl replacement has occurred.

[0288] In embodiments, metal hydroxide (e.g., aluminum hydroxide) binding polypeptides fused to an immunomodulatory polypeptide allow strong binding to aluminum hydroxide. Various immunomodulatory polypeptides fused to ABPs are assessed herein. In embodiments, ABPs adsorbed to aluminum hydroxide in serum and are used to retain proteins and peptides, e.g., within a tumor environment. In embodiments, polypeptides with greater phosphorylation may be retained on alum for longer in serum conditions. In embodiments, the polypeptide,GGGGSFQSEEQQGGGSGGSEEGGMESEESNGGGSGGSEEGGGGSHHHHHH, referred to as “ABP10,” demonstrated phosphorylation with an increase of phosphorylation of 4-6-fold when the protein was expressed with a wild-type (WT) kinase (e.g., WT Fam20C kinase), compared to when the protein was expressed with a mutant kinase (e.g., mutant Fam20C kinase). In embodiments, ABP10 consists of four SXE motifs (e.g., 8 SXE motifs in an antibody expressing two HC or LC fusions), a prevalent phosphorylation site motif, separated by short spacer sequences.Page 72 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0289] In embodiments, the metal hydroxide-binding polypeptide comprises one or more spacer sequences. In embodiments, a spacer sequence allows for high efficiency phosphorylation in comparison to a cognate peptide that lacks the spacer sequence between phosphorylation sites due to, e.g., steric hindrance or charge repulsion from the closeness of phosphorylation sites and / or phosphorylated residues. In embodiments, one or more spacer sequences separate two or more adjacent phosphorylation sites, or are otherwise placed adjacent to or between resid ues / motifs that are targeted for phosphorylation.

[0290] In embodiments, the one or more spacer sequences comprises about or at least about 1 amino acid to about or at least about 20 amino acids, optionally comprising about or at least about 1 amino acid, about or at least about 2 amino acids, about or at least about 3 amino acids, about or at least about 4 amino acids, about or at least about 5 amino acids, about or at least about 6 amino acids, about or at least about 7 amino acids, about or at least about 8 amino acids, about or at least about 9 amino acids, about or at least about 10 amino acids, about or at least about 15 amino acids, or about or at least about 20 amino acids, or any length of amino acids therebetween.

[0291] In embodiments, the one or more spacer sequences comprises glycine, serine, glutamic acid, and / or cysteine residues. In embodiments, the one or more spacer sequences comprises one or more glycine residues, optionally comprising about or at least about 4 glycine residues. In embodiments, the metal hydroxide-binding polypeptide comprises two or more S-X-E phosphorylation sites, and wherein the two or more S-X-E phosphorylation sites are separated by a spacer sequence comprising 4 glycine residues.

[0292] In embodiments, metal-hydroxide binding polypeptides are designed and / or synthesized as described in the published PCT Application, WO2022235755, the entire contents are incorporated herein by reference. In embodiments, metal-hydroxide binding polypeptides are characterized by enhanced metal hydroxide (e.g., alum) retention relative to an appropriate reference (e.g., to ABP10). Additionally, in embodiments, metal-hydroxide binding polypeptides are characterized by improved efficacy, as compared to an appropriate reference (e.g., to ABP10), when administered to a subject with a tumor.

[0293] In embodiments, immunomodulatory moieties (e.g., antibodies, antibody agents and ligands) anchored to a metal hydroxide produce more potent signaling than those without alum anchoring. Without wishing to be bound by theory, the more potent signal is due to an increased localized concentration of the metal hydroxide-binding polypeptide fused protein on the metal hydroxide which is conducive to signaling via TNFRSF family proteins.Page 73 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0294] Thus, among other things, the present disclosure identifies particularly useful immunomodulatory moieties that, when associated with an (e.g., covalently linked to) an ABP and / or adsorbed to aluminum hydroxide, can produce beneficial effects for treatment of a disease (e.g., cancer).

[0295] Additionally, in embodiments, a metal-hydroxide binding polypeptide comprises an amino acid sequence that includes a plurality of phosphorylation sites, so that it can adopt phosphorylated and unphosphorylated forms. In some embodiments, a metal-hydroxide binding polypeptide comprises at least one kinase target motif. A target kinase motif comprises an amino acid that is phosphorylated by a kinase. Amino acids that are typically phosphorylated include a hydroxyl, such as serine (Ser, S), threonine (Thr, T), and tyrosine (Tyr, Y) residues. A kinase motif refers to the amino acid sequence immediately N- and / or C-terminal to the amino acid residue capable of being phosphorylated. Without wishing to be bound by any one theory, many kinases comprise structural features that confer specificity such that the kinase phosphorylates a particular amino acid (e.g., serine, threonine, or tyrosine) of a particular kinase target motif.

[0296] Recognized kinase target motifs are highly diverse depending on the particular type of kinase. In some embodiments, the present disclosure provides metal-hydroxide binding polypeptides comprising one or more kinase target motifs of a secretory pathway kinase. The secretory pathway, which is the pathway by which a cell secretes proteins and / or other biomolecules into the extracellular space, refers to the endoplasmic reticulum (ER), Golgi apparatus (Golgi), cell membrane, and lysosomal storage compartments as well as the vesicles that travel between them. Secretory pathway kinases are localized throughout the secretory pathway (e.g., at the ER, Golgi, etc.) and function to phosphorylate proteins destined for secretion (Sreelatha et al. Biochimica et biophysica acta vol. 1854,10 Pt B (2015): 1687-93).

[0297] In some embodiments, a relevant kinase is a naturally occurring secretory pathway kinase (e.g., is endogenously targeted to the secretory pathway to function). In some embodiments, a secretory pathway kinase comprises a signal sequence that targets the kinase to the secretory pathway. Naturally-occurring human secretory pathway kinases include, for example, four-jointed box kinase 1 , Fam20A, Fam20B, Fam20C, vertebrate lonesome kinase (VLK), SGK196, and Fam69A, Fam69B, and Fam69C.

[0298] In some embodiments, a relevant kinase is a non-naturally occurring secretory pathway kinase. In some embodiments, a non-naturally occurring kinase is produced by linking a secretory signal peptide to a kinase endogenously localized to a non-secretory pathway cellular compartment.

[0299] In some embodiments, a kinase target motif is a target kinase motif of a secretory pathway kinase. In some embodiments, a secretory pathway kinase target kinase motif comprises an S-X-E motif. For example, Page 74 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005Fam20C phosphorylates serine and has been shown to phosphorylate kinase targets motif comprising the amino acid sequence Ser-X-Glu (e.g., S-X-E), Ser-X-pSer (e.g., S-X-pS), and Ser-X-GIn-X-X-Asp-Glu-Glu (S- X-Q-X-X-D-E-E, SEQ ID NO: 364) wherein X is any amino acid, and pS is phosphorylated serine (Mercier, et al (1981) Biochimie, 63: 1-17; Mercier et al (1971) Eur J. Biochem. 23:41-51 ; Lasa-Benito (1996) FEES Lett. 382:149; Brunati, et al (2000) 3:765, Tagliabracci, et al (2015) Cell 161 :1619-1632; Tagliabracci, et al (2012) Science 336:1150-1153). In some embodiments, a target kinase motif comprises the amino acid sequence SEE with an additional residue selected from A, E, and Q. In some embodiments, a target kinase motif comprises the amino acid sequence SEEE (SEQ ID NO: 399). In some embodiments, a target kinase motif comprises the amino acid sequence SEEA (SEQ ID NO: 400). In some embodiments, a target kinase motif comprises the amino acid sequence SEEQ (SEQ ID NO: 401). In some embodiments, a target kinase motif comprises the amino acid sequence SEE.

[0300] In some embodiments, a metal-hydroxide binding polypeptide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs. In some embodiments, a metal- hydroxide binding polypeptide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve S-X-E motifs. In some embodiments, a metal-hydroxide binding polypeptide comprises more than four S-X-E motifs. In some embodiments, a metal-hydroxide binding polypeptide comprises eight S-X-E motifs. In some embodiments, the number of target kinase motifs (e.g., S-X-E motifs) contributes to the number of phosphorylated residues on a metal-hydroxide binding polypeptide.

[0301] In some embodiments, a metal-hydroxide binding polypeptide is a metal-hydroxide binding polypeptide whose amino acid sequence includes a plurality of phosphorylation sites. In some embodiments, a plurality of phosphorylation sites comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs. In some embodiments, a plurality of phosphorylation sites comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve S-X-E motifs. In some embodiments, a plurality of phosphorylation sites comprises more than four S-X-E motifs. In some embodiments, a plurality of phosphorylation sites comprises more than eight S-X-E motifs. In some embodiments, the number of target kinase motifs (e.g., S-X-E motifs) contributes to the number of phosphorylated residues on a metal-hydroxide binding polypeptide.

[0302] In some embodiments, the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs (e.g., S-X-E motifs) are directly adjacent (e.g., linked) to the next target kinase (e.g., S-X-E motif). In some embodiments, the at least one, two, three, four, five, six, seven, eight, nine, ten,Page 75 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 eleven, or twelve target kinase motifs (e.g., S-X-E motifs) are separated (e.g., linked) to the next target kinase motif (e.g., S-X-E motif) by a spacer. In some embodiments, the spacer comprises at least one glycine residue. In some embodiments, the spacer comprises a plurality of glycine residues. In some embodiments, the spacer comprises three glycine residues. In some embodiments, the spacer comprises at least four glycine residues. In some embodiments, the spacer has a sequence that comprises four glycine residues. In some embodiments, the spacer has an amino acid sequence comprising GGGSGGGG (SEQ ID NO: 397). In some embodiments, the spacer has an amino acid sequence comprising GGGEGGGG (SEQ ID NO: 398).

[0303] In some embodiments, a metal-hydroxide binding polypeptide comprises four S-X-E motifs and three spacers comprising four glycine residues. In some embodiments, a metal-hydroxide binding polypeptide comprises six S-X-E motifs and five spacers comprising four glycine residues. In some embodiments, a metal- hydroxide binding polypeptide comprises eight S-X-E motifs and seven spacers comprising four glycine residues. In some embodiments, a metal-hydroxide binding polypeptide comprises four motifs with the amino acid sequence, SEEE (SEQ ID NO: 399), and three spacers comprising three glycine residues. In some embodiments, a metal-hydroxide binding polypeptide comprises four motifs with the amino acid sequence, SEEA (SEQ ID NO: 400), and three spacers comprising three glycine residues. In some embodiments, a metal- hydroxide binding polypeptide comprises four motifs with the amino acid sequence, SEEQ (SEQ ID NO: 401), and three spacers comprising three glycine residues. In some embodiments, a metal-hydroxide binding polypeptide comprises four motifs with the amino acid sequence, SEE, and three spacers comprising the amino acid sequence, GGGSGGGG (SEQ ID NO: 397). In some embodiments, a metal-hydroxide binding polypeptide comprises four motifs with the amino acid sequence, SEE, and three spacers comprising the amino acid sequence, GGGEGGGG (SEQ ID NO: 398).

[0304] In some embodiments, a metal-hydroxide binding polypeptide comprises six S-X-E motifs, wherein each S-X-E motif is directly adjacent to the next S-X-E motif. In some embodiments, a metal-hydroxide binding polypeptide comprises eight S-X-E motifs, wherein each S-X-E motif is directly adjacent to the next S-X-E motif.

[0305] In some embodiments, a metal-hydroxide binding polypeptide comprise an ending sequence (e.g., an amino acid sequence at the c-terminus of the fusion polypeptide. In some embodiments, an ending sequence comprises a plurality of amino acid residues. In some embodiments, a plurality of amino acid residues comprises GGGG (SEQ ID NO: 402). In some such embodiments, an ending sequence comprises the amino acid sequence GGGGS (SEQ ID NO: 214). In some such embodiments, an ending sequencePage 76 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 comprises the amino acid sequence GGGGHHHHHH (SEQ ID NO: 403). In some such embodiments, an ending sequence comprises the amino acid sequence GGGGSHHHHHH (SEQ ID NO: 404). In some embodiments, an ending sequence comprises an optional tag (e.g., hexahistidine, HHHHHH SEQ ID NO: 144), as discussed elsewhere herein.

[0306] In some embodiments, a metal hydroxide binding polypeptide has a sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to an ABP sequence from Table 3. In some embodiments, a metal hydroxide binding polypeptide has a sequence with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to an ABP sequence from Table 3.

[0307] In some embodiments, a metal hydroxide binding polypeptide has a sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 128-143. In some embodiments, a metal hydroxide binding polypeptide has a sequence with at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from SEQ ID NOs: 128-143.

[0308] In some embodiments, a metal hydroxide binding polypeptide is ABP20-G4-GE. In some embodiments, a metal hydroxide binding polypeptide has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 141.

[0309] In some embodiments, a metal hydroxide binding polypeptide is ABP20-G4-8x-GE. In some embodiments, a metal hydroxide binding polypeptide has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to SEQ ID NO: 143.Linkers

[0310] In some embodiments, fusion polypeptides as described herein may include one or more linkers or spacers.

[0311] For example, in some embodiments, fusion polypeptides comprise an immunomodulatory polypeptide comprising a first and a second immune agonist moiety. In some embodiments, a first and a second immune agonist moiety are linked via a first linker. In embodiments, the agonist comprises a linker, e.g., an amino acid linker. In embodiments, agonists herein comprise an amino acid linker between the antibody or antibody-format molecule and the metal hydroxide-binding polypeptide.Page 77 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0312] In some embodiments, fusion polypeptides of the present disclosure comprise an immunomodulatory polypeptide and a metal-hydroxide binding polypeptide. In some embodiments, an immunomodulatory polypeptide and a metal-hydroxide binding polypeptide are linked via a second linker.

[0313] In some embodiments, a first linker and / or a second linker is a polypeptide linker. In some embodiments, a polypeptide linker is synthetic. For example, a synthetic polypeptide linker may comprise non- naturally occurring polypeptides which are modified forms of naturally occurring polypeptides.

[0314] In some embodiments, polypeptide linkers of the present disclosure are at least one amino acid in length and can be any suitable number of amino acids. In some embodiments, a polypeptide linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In embodiments, amino acid linkers of the present disclosure are at least one amino acid in length and comprise any suitable number of amino acids. In embodiments, amino acid linkers herein are about or at least about 1 to about or at least about 50 amino acids in length. In embodiments, the amino acid linker comprises about or at least about 1 amino acid, about or at least about 3 amino acids, about or at least about 5 amino acids, about or at least about 10 amino acids, about or at least about 15 amino acids, about or at least about 20 amino acids, about or at least about 25 amino acids, about or at least about 30 amino acids, about or at least about 35 amino acids, about or at least about 40 amino acids, about or at least about 45 amino acids, or about or at least about 50 amino acids, including lengths therebetween.

[0315] In embodiments, the amino acid linker is synthetic, for example, a synthetic polypeptide linker comprises non-naturally occurring polypeptides which are modified forms of naturally occurring polypeptides. In embodiments, the amino acid linker is rigid (e.g., alpha-helical linkers). In embodiments, the amino acid linker is flexible. In embodiments, the amino acid is cleavable. Non-limiting examples of linkers include, in embodiments, helix-forming peptide linkers (e.g., A(EAAAK)nA SEQ ID NO: 405, where n = 1-5), dipeptide linker (Phe-Lys, LE), thrombin-sensitive disulfide cyclopeptide linker, disulfide linker, and alpha-helix forming linker LEA(EAAAK)nALEA(EAAAK)nALE (SEQ ID NO: 406, wherein n = 1-5). In embodiments, the linker is one that is used with antibody-drug conjugates, such as valine-citrulline based linkers (Val-Cit, Val-Cit-PABC, etc.) hydrazone linker, glutathione-sensitive linkers, and the like.

[0316] In embodiments, the amino acid linker comprises or consists of a glycine and / or serine (Gly-Ser or G-S linker). In embodiments, the Gly-Ser linker comprises or consists of an amino acid sequence of (GlyxSer)y, where x and y are positive integer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In embodiments, x is 4 and y is a number between 1 and 10, inclusive. In embodiments, a Gly-Ser linker is (Gly4Ser)i (SEQ ID NO: 214). In Page 78 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 embodiments, a Gly-Ser linker is (Gly4Ser)2 (SEQ ID NO: 215). In embodiments, a Gly-Ser linker is (Gly4Ser)3 (SEQ ID NO: 216). In embodiments, a Gly-Ser linker is (Gly4Ser)4 (SEQ ID NO: 217). In embodiments, a Gly- Ser linker is (Gly4Ser)s (SEQ ID NO: 407). In embodiments, a Gly-Ser linker is (Gly4Ser)e (SEQ ID NO: 408). In embodiments, a Gly-Ser linker is (Gly4Ser)? (SEQ ID NO: 409). In embodiments, a Gly-Ser linker is (Gly SerJs (SEQ ID NO: 410). In embodiments, a Gly-Ser linker is (Gly4Ser)g (SEQ ID NO: 411). In embodiments, a Gly-Ser linker is (Gly4Ser)io (SEQ ID NO: 412). Other exemplary linkers include GSn linkers, for example having one or more repeats of GGSG (SEQ ID NO: 413), GSAT (SEQ ID NO: 414), SEG, and GGS. In embodiments, the amino acid linker is or comprises one or more repeats of G, S, GG, SS, GS, SG, GGS, SSG, GGGG (SEQ ID NO: 402), SGGG (SEQ ID NO: 415), GGGS (SEQ ID NO: 421), GGGGS (SEQ ID NO: 214), GGGGEGGGG (SEQ ID NO: 416), and GGGGSGGGG (SEQ ID NO: 417). In embodiments, the “one or more” comprises repeats of each individual linker motif or sequence, for example, between about 1 and about 20 repeats.

[0317] In some embodiments, a second linker comprises a polypeptide linker. In some embodiments, a second linker comprises a plurality of glycine residues. In some embodiments, a second linker comprises a polypeptide linker with the amino acid sequence, GGGGSGGGG (SEQ ID NO: 417). In some embodiments, a second linker comprises a polypeptide linker with the amino acid sequence, GGGGEGGGG (SEQ ID NO: 416).

[0318] In embodiments, spacer sequences between two or more phosphorylation sites in the metal hydroxide-binding polypeptide have the sequence of one or more amino acid linker sequences herein.Variants

[0319] In some embodiments, an immunomodulatory polypeptide or a metal-hydroxide-binding polypeptide utilized in accordance with the present disclosure is a variant of a relevant reference polypeptide (e.g., a wild type polypeptide or functional portion thereof).

[0320] In some embodiments, a variant shows at least 70% identity to its reference polypeptide. In some such embodiments, a variant shows at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to its references polypeptide

[0321] In some embodiments, a variant comprises one or more conservative or otherwise non-disruptive modifications (e.g., substitutions, deletions or additions) relative to its reference. In some embodiments, a variant is free of any disruptive modifications (e.g., substitutions, deletions or additions) so that an immunomodulatory polypeptide maintains one or more functional characteristics of the reference. In somePage 79 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 embodiments, maintains means an immunomodulatory polypeptide display comparable activity (e.g., signaling competency or binding) compared to an appropriate reference standard (e.g., a wild-type immunomodulatory polypeptide). For example, in some such embodiments, an immunomodulatory polypeptide maintains at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more activity compared to an appropriate reference standard (e.g., a wild-type immunomodulatory polypeptide).Metal hydroxides

[0322] In some embodiments, the present disclosure provides fusion polypeptides comprising an immunomodulatory polypeptide and a metal-hydroxide binding polypeptides, wherein the fusion polypeptide, when exposed to a metal-hydroxide forms a complex therewith. In embodiments, a complex is formed via adsorption of the fusion polypeptide to a metal-hydroxide. Without wishing to be bound by theory, adsorption of a fusion polypeptide to a metal hydroxide occurs by ligand exchange. Ligand exchange, for example, is a substitution or exchange of a surface hydroxyl by another ligand. In some embodiments, substitution or exchange of a surface hydroxyl occurs by a hydroxyl-replacement group (e.g., a phosphate group).

[0323] In embodiments, anchoring to a metal hydroxide is not necessary (e.g., where a metal hydroxide- binding polypeptide fusion is kept with a phosphorylated polypeptide free and substantially not contact with a metal hydroxide before administration).

[0324] In some embodiments, a metal-hydroxide is a substance that includes at least one hydroxyl group bound to a metal. In accordance with the present disclosure, in some embodiments, a metal-hydroxide can adsorb fusion polypeptides comprising a hydroxyl-replacement moiety. In some embodiments, a hydroxyl- replacement moiety is a phosphate group.

[0325] In some embodiments, a metal-hydroxide is selected based on its inherent qualities or characteristics. In some embodiments, a metal-hydroxide is selected due to its biocompatibility for use in a subject (e.g., a mammal, e.g., a human). For example, in embodiments, the metal hydroxide is the FDA- approved vaccine adjuvant Alhydrogel® (aluminum hydroxide) which forms a spontaneous complex through a phosphoserine mediated ligand exchange reaction with the phosphorylated metal hydroxide-binding polypeptide. In embodiments, agonist / Alhydrogel® complexes are administered intratumorally (e.g., IT), where they are locally retained due to their size and charge, leading to an antibody depot that promotes potent and long-lasting local immune activation. In some embodiments, a metal-hydroxide is aluminum hydroxide (e.g., alum). In some embodiments, a metal-hydroxide is iron-hydroxide. In embodiments, the metal hydroxide is orPage 80 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 comprises one or more of aluminum hydroxide, aluminum phosphate, calcium hydroxide, calcium phosphate, iron hydroxide, magnesium hydroxide, barium hydroxide, calcium hydroxide, zinc hydroxide, and zirconium hydroxide. In embodiments, the metal hydroxide is or comprises aluminum hydroxide. A skilled artisan will recognize any number of metal-hydroxide may be successfully utilized in accordance with the present disclosure.Ta s

[0326] In embodiments, proteins herein comprise one or more purification tags. For example, in embodiments, the purification tag comprises a polyhistidine tag (e.g., 6xHis tag, HHHHHH, SEQ ID NO: 144) fused at the N-terminus or C-terminus of the antibody or antibody-format molecule, or of the metal hydroxide- binding polypeptide. In embodiments, a purification tag is not removed from the protein during production and / or formulation into a pharmaceutical agent. Alternatively, in embodiments, a purification tag is optional and / or removed.

[0327] In some embodiments, a fusion polypeptide of the present disclosure comprises an immunomodulatory polypeptide that comprises an immune agonist moiety and a metal-hydroxide binding polypeptide whose amino acid sequence includes a plurality of phosphorylation sites, so that it can adopt phosphorylated and unphosphorylated forms. In some embodiments, a fusion polypeptide of the present disclosure comprises an ending sequence. In some embodiments, an ending sequences comprises a tag. A tag, as used herein, is an amino acid sequence that, when detected and / or measured in a particular sample, indicates that the protein to which it was linked is present in the sample and can provide a quantitative measurement thereof. A variety of tags known in the art may be used in accordance with the present disclosure. For example, in some embodiments, a tag comprises a FLAG tag, a polyhistidine tag, a V5 tag, a MBP tag.

[0328] In some embodiments, a tag is inserted at the N- or C-terminus of the fusion polypeptide to minimize interference with fusion polypeptide function. In some embodiments, the tags are be placed internally in-frame within the fusion polypeptide sequence without affecting functionality. Preferred locations for the tag may be determined in part based on available empirical data (e.g., from affinity tag or other fusion polypeptide experiments such as polyhistidine, FLAG tag, MBP tag, etc.), three dimensional structures of the fusion polypeptide or similar polypeptides and / or in vivo or in vitro expression experiments.

[0329] In some embodiments, a tag can also contain a short sequence motif, such as an affinity tag or chromatography tag to allow for partial or complete purification from a complex mixture or preparation. In somePage 81 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 embodiments, the peptide tags can be designed to allow inclusion of a fixed number (e.g., one, two, three, or more) of a set of preselected (e.g., one, two, three, or more) amino acids.Production of fusion polypeptides and metal-hydroxide complexes

[0330] In one aspect of the present disclosure, fusion polypeptide-metal hydroxide complexes described herein are produced by (1) making a fusion polypeptide; (2) phosphorylating a fusion polypeptide; and (3) contacting a fusion polypeptide with metal hydroxide. In embodiments, step (3) is optional.

[0331] In some embodiments, fusion polypeptides described herein are made in host cells using techniques for exogenous expression. Methods of exogenously expressing polypeptides are well known in the art and the skilled artisan would recognize a variety of techniques could be successfully utilized.

[0001] Recombinant polynucleotides (e.g., DNA or RNA) encoding for fusion polypeptides of the present disclosure may be prepared by a variety of methods available. In embodiments, the fusion protein and / or kinase for phosphorylating the fusion protein is encoded in a nucleic acid. In embodiments, the nucleic acid molecule is DNA. In embodiments, the DNA is a plasmid, circular DNA, non-viral vector, or viral vector. In embodiments, the viral vector is a lentiviral vector or an adeno-associated viral vector (AAV). In embodiments, the nucleic acid molecule is RNA. In embodiments, the RNA is mRNA, circular RNA, or self-amplifying RNA (saRNA). In embodiments, the nucleic acid is configured for stable cell expression or transient cell expression of the agonist. In embodiments, sequences encoding fusion polypeptides are excised from DNA, for example, using restriction enzymes, and / or are amplified from plasmids or genomic polynucleotide sequences using, for example, polymerase chain reaction, or may be synthesized using chemical synthesis techniques. In some embodiments, a combination of known methods is utilized to prepare a recombinant polynucleotide encoding for fusion polypeptides of the present disclosure.

[0002] In embodiments, the nucleic acid molecule comprises a sequence that facilitates cellular export of the agonist (e.g., an antibody secretion signal). Secretion signals for antibody production are known in the art. In embodiments, the antibody secretion signal or signal peptide is or comprises the amino acid sequence of SEQ I D NOs: 418 or 419, or a sequence having 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, or deletions thereto. In embodiments, the amino acid sequence of SEQ ID NO: 418 is an antibody light chain secretion signal / signal peptide. In embodiments, the amino acid sequence of SEQ ID NO: 419 is an antibody heavy chain secretion signal / signal peptide.Page 82 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0003] In embodiments, the signal peptide is a mammalian signal peptide. In embodiments, the signal peptide is suitable for secretion of an antibody (e.g., native signal peptide). Various signal peptides suitable for secretion for recombinant protein production are described, for example, in US Patent Nos. 5,580,758; 6,107,057; 7,741 ,075; 10,435,694; 11,306,127; 11 ,370,815; US Patent Application Publication Nos. 2007 / 0117186, 2010 / 0055125, 2016 / 0168198, the disclosure of each of which is hereby incorporated by reference. In embodiments, the signal peptide is a signal peptide selected from DNase, alpha mating factor, human serum albumin, alpha amylase, glucoamylase, inulinase, insulin, invertase signal peptide, lysozyme.

[0004] In embodiments, the nucleic acid sequences herein are codon optimized for expression in a host cell. Persons skilled in the art, upon the benefit of this disclosure in its entirety will be familiar with how to alter a DNA / RNA sequence for codon usage preferences of the host cell of choice, and understand how to select the host organism for desired post-translation modification profile (e.g., glycosylation).

[0332] In embodiments, polypeptides described herein (e.g., kinases, antibodies, stabilizing domains, metal hydroxide-binding peptides, purification tags, etc.) are manufactured in transfected host cells using recombinant DNA techniques. In embodiments, a recombinant DNA molecule encoding a polypeptide is prepared. Various appropriate methods of preparing such DNA molecules are known in the art, and those skilled in the art will be familiar with useful such technologies. For instance, in embodiments, sequences coding for a particular polypeptides may be excised from DNA using suitable restriction enzymes. Alternatively or additionally, in embodiments, PCR amplification and / or chemical synthesis technologies are utilized to prepare part or all of a relevant DNA molecule.

[0333] In embodiments, purified nucleic acid molecules are contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced or transfected with the vector. In embodiments, the expression vectors herein contain one or more nucleic acid sequences encoding one or more fusion proteins herein and cells therein are transfected or transduced with one or more of such vectors. In embodiments, the transfected or transduced cells are mammalian cells.

[0334] Recombinant polynucleotides encoding fusion polypeptides of the present disclosure may be cloned into a vector capable of expressing a fusion polypeptide. Cloning may be carried out according to a variety of methods available (e.g., Gibson assembly, restriction digest and ligation, etc.). In some embodiments, a vector is a viral vector. In some embodiments, a vector is a non-viral vector. In some embodiments, a vector is a plasmid.Page 83 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0335] In some embodiments, a vector capable of expression comprises a recombinant polynucleotide that encodes a fusion polypeptide of the present disclosure operatively linked to a sequence or sequences that control expression (e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.). In some embodiments, a sequence or sequences that control expression are selected to achieve a desired level of expression. In some embodiments, more than one sequence that controls expression (e.g., promoters) are utilized. In some embodiments, more than one sequence that controls expression (e.g., promoters) are utilized to achieve a desired level of expression of a plurality of recombinant polynucleotides that encode a plurality polypeptides. In some embodiments, a plurality of recombinant polypeptides are expressed from the same vector (e.g., a bicistronic vector, a tricistronic vector, multicistronic.). In some embodiments, a plurality of recombinant polypeptides are expressed, each of which is expressed from a separate vector.

[0336] Host cells suitable for transfection, transformation, and recombinant protein expression are known. In embodiments, any well-known host cells are suitable for use in the methods disclosed herein. In embodiments, the host cell is suitable for recombinant protein expression. Selection of a particular host is dependent upon a number of factors recognized by the art, which may include in non-limiting embodiments, compatibility with the chosen expression vector, toxicity of the peptides encoded therein, rate of transformation, nucleic acid propagation rates (e.g., plasmid / vector copy number), ease of recovery of the recombinant proteins, secretion of the antibody-format molecules, expression characteristics, glycosylation patterns and glycan content, biosafety, manufacturability, and cost. In non-limiting embodiments, useful microbial hosts include bacteria (e.g., E. coli spp.), yeast (e.g., Saccharomyces spp.), fungi, insects (e.g., SF9 cells and baculoviral expression systems), plants, and mammalian cells (e.g., human, hamster, etc.) in culture, or other hosts known in the art. For example, in non-limiting embodiments, the host cell is a mammalian cell selected from a Chinese hamster ovary (CHO) cell (CHO-K1 , CHO-DHB11 , CHO-DXB1 , CHO-S, CHO-DG44, CHO- M), human embryonic kidney (HEK293, HEK293T) cell, K562 human lymphoblast cell, U2OS human osteosarcoma cell, HeLa cell, Vero cell (Vero, Vero 76, Vero E6), baby hamster kidney (BHK) cell, mouse myeloma cell (NSO, Sp2 / 0), monkey kidney cell (COS, CV1), K562 human lymphoblast cell, human hepatocellular carcinoma cell (Hep G2), primary human fibroblast (human dermal fibroblast, HDFa), human cervical carcinoma cell (HELA, 3T3), myeloma cell, PERc6 cell, or CAP cell.

[0337] In some embodiments, a vector capable of expression comprising a recombinant polynucleotide encoding a fusion polypeptide of the present disclosure is used to express a fusion polypeptide in a host cell.Page 84 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005In embodiments, a suitable host cell may be selected from a variety of the available and known host cells (e.g., Human Embryonic Kidney (HEK) cells, suspension HEK293 cells, Chinese Hamster Ovary cells) suitable expressing fusion polypeptides disclosed herein.

[0338] A variety of methods are available to introduce a vector into host cells. In some embodiments, a vector may be introduced into host cells using transfection. In some embodiments, transfection is completed, for example, using calcium phosphate transfection, lipofection, or polyethylenimine-mediated transfection. In some embodiments, a vector may be introduced into a host cell using transduction.

[0339] In some embodiments, a transformed host cells are cultured following introduction of a vector into a host cell to allow for expression of said recombinant polynucleotides. In some embodiments, a transformed host cells are cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours or longer. Transformed host cells are cultured in growth conditions (e.g., temperature, carbon-dioxide levels, growth medium) in accordance with the requirements of a host cell selected. A skilled artisan would recognize culture conditions for host cells selected are well known in the art.

[0340] In embodiments, described herein is a composition of a clonal cell population for, e.g., stable protein expression. In embodiments, cell populations herein (and compositions thereof) are suspended in one or more excipients, e.g., such as cell media (e.g., MEM, DMEM, RPMI 1640, Ham’s F-12K, Ham’s F-10, GMEM, BME, McCoy's 5A, M200, Leibovitz's L-15, or IMDM), fetal bovine serum (FBS), amino acids (L- glutamate, non-essential amino acids (NEAA)), sodiumpyruvate, pH buffer (HEPES, etc.), NaCI, pH indicator (e.g., phenol red), hydrocortisone, dexamethasone, cell factors, (e.g., epidermal growth factor, insulin, etc.). In embodiments, cell populations herein (and compositions thereof) are suspended in one or more cryoprotectant to protect cells for freeze-thaw cycles, such as DMSO, glycerol, sucrose, ethylene glycol (EG), propylene glycol (PG), and polyethylene glycol (PEG).

[0341] In embodiments, nucleic acids herein (and compositions thereof) are lyophilized, or suspended in one or more excipients, including nuclease-free water, Tris buffer, salt (e.g., magnesium chloride), etc.Phosphorylating fusion polypeptides

[0342] In some embodiments, fusion polypeptides described herein are phosphorylated by contacting a fusion polypeptide with a kinase. In some embodiments, a fusion polypeptide is contacted with a kinase by coexpressing a fusion polypeptide in a host cell with a kinase. In some embodiments, co-expression is achieved Page 85 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 by introducing two vectors, one comprising a recombinant polynucleotide encoding a fusion polypeptide and one comprising a recombinant polynucleotide encoding a kinase, into a host cell. In some embodiments, coexpression is achieved by introducing a single, multicistronic (e.g., bicistronic) vector that comprises a plurality of recombinant polynucleotides. In some embodiments, a recombinant polynucleotides encode a fusion polypeptide and a recombinant polynucleotide encoding a kinase. In some embodiments, a transformed host cell is cultured following introduction of a vector into a host cell. Without wishing to be bound by any one theory, upon co-expression of a fusion polypeptide and a kinase in a host cell, a kinase can contact a fusion polypeptide, phosphorylating it.

[0343] In some embodiments, co-expression is achieved by introducing two vectors, one comprising a recombinant polynucleotide encoding a fusion polypeptide and one comprising a recombinant polynucleotide encoding a kinase, into a host cell. In some embodiments, two vectors are introduced at a ratio of vector encoding fusion polypeptide to vector encoding a kinase introduced into a host cell optimized to achieve a desired, relative level of expression of fusion polypeptide to kinase. In some embodiments, a ratio of vector encoding fusion polypeptide to vector encoding a kinase is 1 :1, 2:1, 3:1, 4:1, 5:1 , 6:1 , 7:1, 8:1 , 9:1, 10:1 , 11 :1 , 12:1 , 13:1, 14:1 , 15:1 , 16:1, 17:1 , 18:1 , 19:1 , 20:1 , 30: 1 , 40:1 , 50:1 , 60:1 , 70:1 , 80:1 , 90:1, or 100:1.

[0344] In some embodiments, co-expression is achieved by introducing one vector comprising both a recombinant polynucleotide encoding a fusion polypeptide and a recombinant polypeptide encoding a kinase (e.g., a bicistronic vector) into a host cell. In some embodiments, a recombinant polynucleotide encoding a fusion polypeptide and a recombinant polynucleotide encoding a kinase are operatively linked to a sequence or sequences that control expression (e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.). In some embodiments, a sequence or sequences that control expression are selected to achieve a desired level of expression. In some embodiments, multiple sequences that control expression (e.g., promoters) are utilized to achieve a desired ratio of expression of fusion polypeptide to kinase. In some embodiments, a ratio is 1 :1 , 2:1 , 3:1 , 4:1 , 5:1 , 6:1, 7:1 , 8:1 , 9:1 , 10:1 , 11 :1 , 12:1, 13:1 , 14:1 , 15:1 , 16:1 , 17:1, 18:1 , 19:1 , 20:1, 30: 1 , 40:1, 50:1 , 60:1 , 70:1 , 80:1 , 90:1, or 100:1.

[0345] In embodiments, methods of phosphorylating proteins herein utilize a kinase to phosphorylate a co-expressed agonist. In embodiments, the kinase is selected from Fam20C, protein kinase A (PKA), cAMP- dependent protein kinase, cyclin-dependent kinase, extracellular-regulated kinase-2 (ERK2), casein kinase 1 (CK1), casein kinase 2 (CK2), glycogen synthase kinase-3, calmodulin-dependent protein kinase-2 (CaMK2), Abelson murine leukemia virus tyrosine kinase (ABL), epidermal growth factor receptor (EGFR), rous sarcomaPage 86 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 virus tyrosine kinase (Src), insulin receptor tyrosine kinase (IPK), protein kinase B (PKB / AKT), protein kinase D (PKD), proviral integration site kinase 1-3 (PIM1-3), AMP-activated protein kinase, mitogen-activated protein kinase, and NimA-related kinase.

[0346] In embodiments, the kinase is a Fam20 family kinase, e.g., a Fam20C kinase. In embodiments, the Fam20C kinase comprises an amino acid sequence as described herein.

[0347] In embodiments, methods for producing phosphorylated agonists comprise using one or more nucleic acid molecules encoding the agonist and one or more nucleic acid molecules encoding one or more kinases. In embodiments, a first nucleic acid molecule and a second nucleic acid molecule are in cis with one another, and each is under the control of an operably linked regulatory nucleic acid sequence that controls expression of the agonist and the kinase. In embodiments, the regulatory nucleotide sequence is or comprises one or more of a promoter, activator, enhancer, operator, cap signal, termination sequence, start codon, stop codon, and polyadenylation (polyA) signal. In embodiments, two or more promoters of different strength are used to produce a ratio of the co-expressed agonist and the kinase. For exmaple, in embodiments, a first nucleic acid sequence encoding the agonist sequence is under the control of a CMV promoter or EF1 a promoter. In embodiments, a second nucleic acid sequence encoding the kinase is under the control of a SV40 promoter or Ubc promoter.Complex formation

[0348] In some embodiments, a fusion polypeptide described herein, when exposed to a metal-hydroxide (e.g., aluminum hydroxide, aluminum phosphate, calcium hydroxide, calcium phosphate, iron hydroxide, magnesium hydroxide, barium hydroxide, calcium hydroxide, zinc hydroxide, and / or zirconium hydroxide) forms a complex therewith. In some embodiments, fusion polypeptides comprise hydroxyl replacement groups (e.g., phosphate groups) for adsorption via ligand exchange with a metal hydroxide. In some embodiments, a fusion polypeptide can, via electrostatic interactions, form a complex with a metal hydroxide.

[0349] In some embodiments, a fusion polypeptide metal-hydroxide complex of the present disclosure is formed by mixing. In some embodiments, mixing occurs in a buffer (e.g., a tris-buffered saline buffer). In some such embodiments, a buffer does not contain phosphate. In some such embodiments, a buffer does not contain a substance or substances that solubilize a metal-hydroxide (e.g., citric acid, malic acid, or lactic acid). Without wishing to be bound by any one theory, buffers that contain phosphate compete with, and can hinder, complex formation. In some embodiments, mixing occurs for a duration of time at a particular temperature. In some such embodiments, a duration of time is 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 Page 87 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 minutes, 35 minutes, 40 minutes, or 45 minutes. In some such embodiments, a particular temperature is approximately 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, or 35°C.

[0350] In non-limiting embodiments, exemplary reaction conditions to form the fusion protein metal hydroxide complex comprise incubating the fusion protein with a 10-fold mass excess of metal hydroxide (e.g., aluminum hydroxide) as defined by metal mass. In embodiments, elution is achieved using an elution buffer comprising about or at least about 1 mM to about or at least about 50 mM phosphate (e.g., 5 mM phosphate).Purification

[0351] In embodiments, commonly aberrant products (e.g., residual protein, host cell contaminants, etc.) are removed from fusion polypeptide preparations. A variety of purification technologies are available and known to the skilled artisan.

[0352] In some embodiments, a fusion polypeptide can be purified by a chromatography method. In some embodiments, such a chromatography purification method can be performed with a chromatography method known in the art, including, but not limited to, high-performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, wherein components of a mixture travel through a stationary phase at different speeds, resulting in separation from one another. It will be apparent to a skilled artisan a variety of solid substrates may be used (e.g., beads, particles, microspheres, resins, etc.). For example, in some embodiments, a solid substrate has properties such that, in accordance with the present disclosure, permits a different retention time for a fusion polypeptide relative to any other undesirable components in the preparation of fusion polypeptide.

[0353] In some embodiments, a fusion polypeptide can be purified by an affinity-based purification method. In some embodiments, such an affinity-based purification method may be performed with a solid substrate known in the art. A wide variety of substrates could be utilized, such as, for example, membranes, polymeric beads, magnetic beads, or composites. For example, in some embodiments, a solid substrate (e.g., polymeric beads or particles) coated or pre-charged with a substance or composition (e.g., nickel ions) that has a high binding affinity for a fusion polypeptides can be useful in accordance with the present disclosure such that a fusion polypeptide will bind to a solid substrate, while any other undesirable components present in a preparation will remain in solution. In some embodiments, a fusion polypeptide may be eluted from a solid substrate. In some embodiments, elution may be carried out using specific elution. For example, in somePage 88 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 embodiments specific elution is completed by challenging a polypeptide-substrate complex by an agent or agents that will compete for complexation with either a substrate or a polypeptide, releasing a polypeptide into solution. In some embodiments, elution may be carried out using non-specific elution. For example, in some embodiments, non-specific elution is completed by manipulating solvent or buffer conditions (e.g., increasing concentration of a buffer, e.g., an imidazole buffer) to reduce the associate rate constant, resulting in dissociation of the polypeptide from the substrate.

[0354] In embodiments, the presents of the metal binding polypeptide is useful for purification (e.g., negative charge of phosphorylation). In embodiments, the purification comprises affinity chromatography (e.g., NiNTA chromatography, for example with a histidine tag (hexahistidine)). In embodiments, the purification comprises anion exchange chromatography. In embodiments, the purification comprises densitometric or centrifugal separation by reacting with metal hydroxide particles and then pulling down the reacted protein and metal hydroxide complexes that form.

[0355] In embodiments, methods or producing and / or purifying proteins herein further comprise harvesting cell media used to culture the host cell (e.g., where the agonist is recovered from the cell media). In embodiments, methods of recovering proteins from the cell media include the use of secretion signal peptides, as known in the art and / or described herein, for example, protein expression with a secretion signal and using centrifugation and / or filtration (e.g., 0.45 pm filter) to isolate a sample from the cell media. In embodiments, methods of protein manufacture herein include lysing the host cells to generate a host cell extract (e.g., to purify protein from the host cell lysate). In embodiments, cell lysis is achievable using a variety of well-known methods, such as sonication, homogenization, extrusion, freeze-thaw cycling, chemical and detergent-based lysis, enzymatic lysis, and use of hypotonic or hypertonic lysis buffers.

[0356] In embodiments, purifying the agonist from the cell media or the cell extract is done without using an affinity chromatography step (although such steps can be used). In embodiments, methods of purification herein rely on the high phosphorylation state of the agonist does not rely on affinity chromatography steps. Alternatively, affinity chromatography is used (e.g., a hexahistidine tag, SEQ ID NO: 144, and NiNTA), or for example, Protein A purification to capture antibody-based proteins (e.g., having an Fc domain). In embodiments, an affinity chromatography step includes NiNTA, Protein A resin, maltose-binding protein resin, and the like, which utilizes capture via affinity with a specific amino acid motif / sequence fused onto the protein- of-interest. In embodiments, a protease sequence is used between the protein and the purification tag, e.g., toPage 89 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 remove the tag after purification. Such protease sites are known in the art (e.g, thrombin, viral proteases such as TEV protease, viral 3C protease, etc.). In embodiments, purification tag removal is not necessary.

[0357] In embodiments, the purifying includes one or more purification steps selected from an anion chromatography step, a hydrophobic interaction chromatography step, and a size exclusion chromatography. In embodiments, the purification comprises at least one anionic chromatography capture step and at least one hydrophobic interaction chromatography step. In embodiments, the purification comprises at least two anionic chromatography capture steps and at least one hydrophobic interaction chromatography step. In embodiments, the purification comprises a first anionic chromatography step, a hydrophobic interaction chromatography step, and a second anionic chromatography step. In embodiments, the one or more purification steps comprises an ultrafiltration / diafiltration step (e.g, for buffer exchange and / or protein concentration). In embodiments, a first step of filtration is performed after a hydrophobic interaction chromatography step or after an anionic chromatography capture step. In embodiments, the anionic chromatography capture step utilizes low salt concentrations for binding phosphorylated proteins to a chromatography column and higher salt concentrations to elute the phosphorylated proteins. In embodiments, a high salt concentration for anion exchange elution is about or at least about 300 mM NaCI to about or at least about 1 M NaCI, including concentrations therebetween. In embodiments, the hydrophobic exchange chromatography step utilizes high ammonium sulfate concentrations for binding phosphorylated proteins to a chromatography column and lower ammonium sulfate concentrations to elute phosphorylated proteins. In embodiments, a high ammonium sulfate concentrations for hydrophobic exchange elution is about or at least about 500 mM ammonium sulfate to about or at least about 2 M ammonium sulfate, including concentrations therebetween. In embodiments, the chromatography, e.g., anion chromatography capture step and / or hydrophobic interaction chromatography step, is performed at a pH within the range of about 6 to about 9 (e.g., about 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, or 9), with a flow rate of about or at least about O.1 mL / min to about or at least about 150 mL / min (e.g, depending upon the scale of the purification, size of the column, etc.).

[0358] In embodiments, the method further comprises a viral inactivation step. In embodiments, the viral inactivation step is performed by contacting the recovered cell media used to culture the host cell and / or the cell lysate / extract with a detergent (e.g, Triton® X-100, sodium dodecyl sulfate (SDS), and polysorbate 80).

[0359] In embodiments, the method further comprises introducing one or more nucleic acid molecules into the host cell (e.g, for recombinant protein production and / or kinase-targeted phosphorylation). InPage 90 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 embodiments, the introduction of nucleic acid comprises using one or more of a lipid-based transfection reagent (cationic lipid-based reagent), diethylaminoethyl (DEAE)-dextran, liposome, electroporation, sonoporation, chemical reagent (calcium phosphate), microinjection, or via a non-integrating viral vector (AAV, lentivirus). In embodiments, the method includes transforming a host cell, culturing into a population of transformed host cells, and purifying / selecting for host cells containing the correct expression protein for making the phosphorylated protein. In embodiments, the host cell is cultured using a selectable marker and a selectable marker gene in an expression cassette with the vector / plasmid encoding the CD40 agonist and / or kinase. In non-limiting embodiments, exemplary selectable markers and resistance genes include neomycin- resistance genes, puromycin-resistance genes, bleomycin-resistance genes, hygromycin-resistance genes, or blasticidin-resistance genes.

[0360] In embodiments, host cells are cultured under conventional fermentation conditions so that a desired polypeptide is expressed. Such fermentation conditions are well known in the art. In embodiments, produced polypeptides are purified from culture by methods well known in the art.

[0361] In embodiments, viral vectors are suitable for expression, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors. The choice of viral vectors is informed, in some instances, by the size of a vector, identity of a host cell, need for either a transient or a stable incorporation of a nucleic acid into a host cell, viral titer, etc.

[0362] In embodiments, the one or more nucleic acid molecules for producing phosphorylated agonists comprises one or more nucleic acid molecule encoding the agonist and one or more nucleic acid molecules encoding one or more kinases. In embodiments, a first nucleic acid molecule and a second nucleic acid molecule are in cis with one another, and each is under the control of an operably linked regulatory nucleic acid sequence that controls expression of the agonist and the kinase. In embodiments, the regulatory nucleotide sequence is or comprises one or more of a promoter, activator, enhancer, operator, cap signal, termination sequence, start codon, stop codon, and polyadenylation (polyA) signal. In embodiments, two or more promoters of different strength are used to produce a ratio of the co-expressed agonist and the kinase. In embodiments, a first nucleic acid sequence encoding the agonist sequence is under the control of a CMV promoter or EF1a promoter. In embodiments, a second nucleic acid sequence encoding the kinase is under the control of a SV40 promoter or Ubc promoter.Page 91 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0363] Persons skilled in the art, with the benefit of this disclosure in its entirety, will be aware of the various host cells and expression systems useful for manufacturing the CD40 agonists described herein, as well as methods of testing their purity and activity.Characterization

[0364] Among other things, in some embodiments, the present disclosure provides technologies for characterizing fusion polypeptides (e.g., phosphorylated or unphosphorylated preparations thereof) and / or of complexes comprising such fusion polypeptides and metal hydroxides. In characterization is performed during and / or following production process. In some embodiments, such characterization may involve assessment of one or more of degree of phosphorylation, signaling activity, and / or efficacy.

[0365] In embodiments, methods or producing and / or purifying agonists herein comprises assessing a degree of phosphorylation of the agonist. This assessment, in embodiments, includes using one or more of a malachite green assay, analytical ion exchange, spectrophotometric analysis, colorimetric assay, mass spectroscopy analysis, SDS-PAGE, and Western blotting.

[0366] In embodiments, the degree of phosphorylation (e.g., of agonists herein) is characterized using a variety of methods. For example, in embodiments, the degree of phosphorylation is determined by a colorimetric method. In embodiments, the colorimetric method is or comprises a malachite green assay. In embodiments, a malachite green assay is based on quantification of a green complex formed between Malachite green, molybdate, and free orthophosphate which is measured (e.g., using a spectrophotometer or plate reader). In embodiments, the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) ranges from about 0.5 to about 20 phosphates per agonist (e.g., antibodies and antibody-format molecules having at least one copy of the metal hydroxide-binding polypeptide per heavy / light chain or heavy / light variable domain, for example, exhibiting two times the number of phosphates per molecule for having two chains per agonist). In embodiments, the average number of phosphate ranges from about 0.5- 1 , about 0.5-2, about 0.5-3, about 0.5-4, about 0.5-5, about 0.5-6, about 0.5-7, about 0.5-8, about 0.5-9, about 0.5-10, about 1-2, about 1-3, about 1-4, about 1-5, about 1-6, about 1-7, about 1-8, about 1-9, about 1-10, about 2-3, about 2-4, about 2-5, about 2-6, about 2-7, about 2-8, about 2-9, about 2-10, about 3-4, about 3-5, about 3-6, about 3-7, about 3-8, about 3-9, about 3-10, about 4-5, about 4-6, about 4-7, about 4-8, about 4-9, about 4-10, about 5-6, about 5-7, about 5-8, about 5-9, about 5-10, about 6-7, about 6-8, about 6-9, about 6- 10, about 7-8, about 7-9, about 7-10, about 8-9, about 8-10, to about 9-10. In embodiments, the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is 3, 3.1 , 3.2, 3.3, 3.4, Page 92 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-50053.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7. 7.8, 7.9, 7.10, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.

[0367] In embodiments, agonists, as described herein, are characterized for activity (e.g., binding activity, signaling activity, and / or in vivo anti-tumor, anti-cancer, abscopal, and / or agonism efficacy). In embodiments, activity is characterized by assessing signaling or binding activity (e.g., signaling competency) via CD40 / CD40L signaling axis as compared to an appropriate reference standard (e.g., anti-CD40 compared against CD40L).

[0368] A variety of methods are available to assess signaling competency (e.g., enzyme-linked immunosorbent assay (ELISA), cell surface binding measured by fluorescence activated cell sorting (FACS), isothermal titration calorimetry (ITC), biolayer interferometry (BLI), surface plasmon resonance (SPR), etc.). In embodiments, agonists herein are assessed using an in vitro and / or in vivo assay. In embodiments, the in vivo assay is an animal cancer model and the efficacy is measured by one or more of tumor volume, number of metastasis, survival, and / or abscopal effects (e.g., bilateral tumor model), e.g., as compared to a control population receiving a standard-of-treatment therapy (e.g., one or more of an anti-PD-1 antibody, checkpoint inhibitor (CPI), cisplatin, rapamycin, radiation treatment, etc.) or a control population not receiving a control treatment, or receiving a vehicle control.

[0369] In embodiments, signaling activity is assessed with an in vitro activity assay. In embodiments, an in vitro activity assay comprises measuring activation or inhibition of downstream signaling of CD40 / CD40L from using the agonist. In embodiments, measuring activation or inhibition of downstream activity comprises use of a reporter or a reporter cell line (e.g., a reporter assay), which presents or expresses functional components of one or more signaling pathways to measure pathway agonism. In embodiments, the reporter assay measures activity using a detectable molecule (e.g., a reporter) that correlates with the agonist binding to CD40, and / or anticancer effects of the agonist.Exemplary characterization of phosphate content

[0370] In some embodiments, degree of phosphorylation (e.g., of fusion polypeptides of the present disclosure and / or preparations thereof) is characterized. A variety of methods are available for measurement of degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide). For example, in some embodiments, degree of phosphorylation can be determined by a colorimetric method. In some embodiments, a colorimetric method is or comprises a malachite green assay. Without wishing to be bound by Page 93 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 any one theory, a malachite green assay is based on quantification of a green complex formed between Malachite green, molybdate, and free orthophosphate which can be measured e.g., using a spectrophotometer or plate reader).

[0371] In some embodiments, degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is 0.5-7, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 0.5-6, 0.5-5, 0.5-4, 1-6, 2-6, 3-6, 4-6, 5-6, 0.5- 10, 0.5-9, 0.5-8, 1-10, 1-9, 1-8, 2-10, 2-9, 2-8, 3-10, 3-9, 3-8, 4-10, 4-9, 4-8, 5-10, 5-9, 5-8, 6-10, 6-9, or 6-8. In some embodiments, degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7. 7.8, 7.9, 7.10, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.

[0372] In some embodiments, the degree of phosphorylation of a polypeptide is measured according to a variety of methods available in the art. In some embodiments, for example and without limitation, the degree of phosphorylation of a polypeptide is measured using a malachite green assay, analytical ion exchange, spectrophotometer, colorimetric assays, and / or western blot.Exemplary characterization of signaling activity

[0373] In some embodiments, fusion polypeptides (and / or complexes thereof) as described herein are characterized for activity (e.g., signaling activity). In some embodiments, activity is characterized by assessing signaling activity (e.g., signaling competency) compared to an appropriate reference standard. An appropriate reference standard can be, for example, a wild-type polypeptide and / or a fusion polypeptide lacking a metal- hydroxide binding polypeptide.

[0374] A variety of methods are available to assess signaling competency. In some embodiments, for example, signaling competency is assessed using an in vitro- or in vivo-based activity assay.

[0375] In some embodiments, signaling activity is assessed with an in vitro activity assay. In some embodiments, an in vitro activity assay comprises measuring activation or inhibition of downstream signaling of a fusion polypeptide. In some embodiments, measuring activation or inhibition of downstream activity comprises use of a reporter (e.g., a reporter assay). In some embodiments, a reporter assay measures activity using a detectable molecule (e.g., a reporter) that correlates with fusion polypeptide activity.Page 94 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0376] In some embodiments, a reporter comprises a fluorescent, bioluminescent, and / or other detectable probe known to those of skill in the art. In some embodiments, a reporter comprises use of a gene reporter. A gene reporter, for example, can be activated upon signaling elicited from a polypeptide. For example, upon activation of gene reporter transcription, a detectable product or enzyme that can be activated upon addition of substrate, generating a detectable product and / or by-product, can be utilized. In some embodiments, an enzyme useful in accordance with a reporter assay is, for example, luciferase or an alkaline phosphatase (e.g, secreted alkaline phosphatase, SEAP). In some such embodiments, a CD40 Effector Cell reporter assay is utilized.

[0377] In some embodiments, signaling activity is assessed with an in vivo activity assay. In some embodiments, a fusion polypeptide is administered to a subject (e.g., a mouse, non-human primate, human, etc.) and activity is assessed. In some embodiments, activity is assessed, for example, by measuring activation or inhibition of downstream signaling of a fusion polypeptide as compared to an appropriate reference standard (e.g, activity of a wild-type polypeptide). A variety of methods are available to measure activation or inhibition of downstream signaling of a fusion polypeptide. For example, and without limitation, differential gene expression, protein expression, and / or alterations in post-translational modifications induced by a fusion polypeptide can be measured.Exemplary efficacy characterization

[0378] In some embodiments, efficacy can be characterized according to a variety of methods that are available. In some embodiments, for example, a fusion polypeptide (or complex thereof) is administered (e.g, by intratumoral or peritumoral injection) to a subject (e.g, mouse, non-human primate, human, etc.) and efficacy is determined in comparison to an appropriate reference standard. An appropriate reference standard can be, for example, a wild-type polypeptide and / or a polypeptide lacking a metal-hydroxide binding polypeptide, or having a metal-hydroxide binding polypeptide in a non-binding (e.g, non-phosphorylated) state.

[0379] In some embodiments, efficacy is determined pre-clinically in an animal model (e.g, in mice, rats, non-human primates, etc.). In some embodiments, a fusion polypeptide is administered (e.g, by intratumoral or peritumoral injection) to an animal model. For example, in some embodiments, an animal model is an animal model with a tumor (e.g, an animal model of cancer). In some embodiments, a cancer animal model is generated by inoculating said animal model with tumor cells. In some embodiments, an animal model is inoculated with tumor cells at the flank region. In some embodiments, an animal model is inoculated with tumor cells in a clinically relevant region (e.g, a mammary fat pad).Page 95 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0380] In some embodiments, an animal model of cancer is administered a fusion polypeptide of the present disclosure. In some embodiments, an animal model of cancer is administered a reference standard (e.g., a wild-type polypeptide and / or a polypeptide lacking a metal-hydroxide binding polypeptide). In some embodiment, a variety of available, pre-determined measurements for efficacy known in the art, such as, for example, tumor volume and / or percent survival are assessed over time relative to an appropriate reference standard (e.g., a wild-type polypeptide and / or a polypeptide lacking a metal-hydroxide binding polypeptide).

[0381] In some embodiments, efficacy of a fusion polypeptide is determined clinically. In some embodiments, a fusion polypeptide is administered (e.g., by intratumoral, peritumoral injection, or into a tumordraining lymph node) to a subject with a tumor. In some embodiments, a variety of available, pre-determined measurements for efficacy known in the art, such as, for example, tumor volume and / or percent survival are assessed over time relative to a subject with a tumor administered reference standard (e.g., a treatment in the art of known efficacy and / or placebo).Compositions

[0382] In some embodiments, the present disclosure, among other things, provides fusion polypeptide preparations. Fusion polypeptide preparations are preparations comprising a fusion polypeptide that, in some embodiments, is purified from a cell culture production of said fusion polypeptide.

[0383] In some embodiments, a fusion polypeptide preparation is a preparation comprising pharmaceutical-grade fusion polypeptide. In some embodiments, a fusion polypeptide preparation is a preparation comprising fusion polypeptide which its one or more characterization attributes are assessed and determined to meet a release and / or acceptance criteria. Examples of such product quality attributes include, but are not limited to, degree of phosphorylation.

[0384] In some embodiments, a fusion polypeptide preparation comprises a phosphorylated form of a fusion polypeptide and a kinase used to phosphorylate a fusion polypeptide (e.g., as described herein). In some embodiments, a kinase is removed from a fusion polypeptide preparation.

[0385] In some embodiments, a phosphorylated fusion polypeptide preparation comprises fusion polypeptides with varying degrees of phosphorylation. In some embodiments, the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) of a preparation of fusion polypeptide is 0.5-7, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 0.5-6, 0.5-5, 0.5-4, 1-6, 2-6, 3-6, 4-6, 5-6, 0.5-10, 0.5-9, 0.5-8, 1-10, 1-9, 1- 8, 2-10, 2-9, 2-8, 3-10, 3-9, 3-8, 4-10, 4-9, 4-8, 5-10, 5-9, 5-8, 6-10, 6-9, or 6-8. In some embodiments, thePage 96 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) of a preparation of fusion polypeptide is 3, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7. 7.8, 7.9, 7.10, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.

[0386] In some embodiments, the present disclosure, among other things, provides a fusion polypeptide preparation comprising a fusion polypeptide-metal hydroxide complex. In some embodiments, a preparation comprising a fusion polypeptide-metal hydroxide complex comprises any of a variety of suitable metal- hydroxide known in the art. For example, and without limitation, a metal-hydroxide may be an aluminum hydroxide or an iron hydroxide. In some embodiments, a fusion polypeptide-metal hydroxide complex comprises a mass ratio of fusion polypeptide to metal hydroxide (e.g., aluminum hydroxide), for example as defined by metal (e.g., aluminum) mass. In some embodiments, the ratio, is 1 :1, 1 :2, 1 :3, 1:4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :11 , 1 :12, 1 :13, 1 :14, 1 :15, 1 :16, 1 :17, 1 :18, 1 :19, or 1:20.

[0387] In some embodiments, the present disclosure, among other things, provides a pharmaceutical composition comprising a fusion polypeptide disclosed herein. In some embodiments, the pharmaceutical composition is formulated as a fusion polypeptide-metal hydroxide complex. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0388] In some embodiments, acceptable pharmaceutical composition formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In some embodiments, formulation material(s) are for subcutaneous and / or intravenous administration. In some embodiments, formulation material(s) are for local administration (e.g., intratumoral or peritumoral administration). In some embodiments, a pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of fusion polypeptides. In some embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides;Page 97 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company (1995). In some embodiments, a pharmaceutical composition comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCI; and / or 10 mM NAOAC, pH 5.2, 9% Sucrose. In some embodiments, an optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In some embodiments, such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of a fusion polypeptide-metal hydroxide complex.

[0389] In some embodiments, a primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in some embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In some embodiments, the saline comprises isotonic phosphate-buffered saline. In some embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In some embodiments, pharmaceutical compositions comprise T ris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute thereof. In some embodiments, a composition comprising a fusion polypeptide metal hydroxide complex or a fusion polypeptide is prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in some embodiments, a composition comprising a fusion polypeptide-metal hydroxide complex or a fusion polypeptide is formulated as a lyophilizate using appropriate excipients such as sucrose.Page 98 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005Uses and Methods of treatment

[0390] In one aspect, the present disclosure relates to methods of treating a subject with a medical condition. In some embodiments, the present disclosure relates to methods of treating a subject with a tumor (e.g., a subject with cancer). In general, methods of treatment are aimed at reducing tumor volume, reducing and / or preventing metastases, prolonging survival, and / or curing the condition. Appropriate subjects or individuals receiving a fusion polypeptide or fusion polypeptide metal hydroxide complex of the present disclosure include, for example, humans or other mammals (e.g., mice, rats, rabbits, dogs, horses, cats, pigs, or non-human primates) that have a tumor (e.g., cancer).

[0391] In embodiments, compositions herein are effective inducers of abscopal effect. In embodiments, the abscopal effect is a phenomenon where localized cancer treatment, like radiation therapy or intratumoral injection, causes shrinkage or regression of tumors not directly treated by the therapy, even at distant locations in the body. Without wishing to be bound by theory, the abscopal effect is an immune-mediated response, where the initial treatment stimulates the body's immune system to attack cancer cells throughout the body, including those outside the treated area.

[0392] In embodiments, described herein are methods of treating cancer using pharmaceutical compositions of the agonists described herein. In embodiments, the method of treatment includes treating a cancer having tumor cells expressing CD40. In embodiments, the method of treatment include treating a cancer that has low or no detectable expression of CD40.

[0393] In embodiments, the agonists disclosed herein function as a CD40 agonist. In embodiments, CD40 agonists, such as agonistic anti-CD40 antibodies and antibody-format molecules, function as a cancer immunotherapy by stimulating the immune system to attack and eliminate cancer cells. In embodiments, the agonists herein function as agonists by activating antigen-presenting cells (APCs), like DCs, which are crucial for initiating and directing T cell responses against tumors. In embodiments, the agonists herein function by immune cell activation (e.g., T cell stimulation), and / or modification of the tumor microenvironment (e.g., altering tumor infiltration by immune cells to make tumor cells more susceptible to immune attack and elimination).

[0394] In embodiments, agonists disclosed herein comprise an anti-CD40 antibody or antibody-format molecule. In embodiments, the anti-CD40 agonists mimicking CD40L by binding to a CD40 receptor expressed by antigen-presenting cells (APCs). In embodiments, APCs that express CD40 are or comprise macrophages,Page 99 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005B cells, T cells, and / or dendritic cells (DCs). In embodiments, the anti-CD40 binding competes for binding with CD40L. In embodiments, this competition for binding is with CD40L expressed by T cells (e.g., CD4+ T-cells). In embodiments, the anti-CD40 agonists bind to a CD40 receptor expressed by one or more cancer cells. The cancer cell, in embodiments, includes one or more of a tumor cell, a cancer stem cell, a circulating tumor cell (CTC), cell belonging to a metastasis, and the like.

[0395] Cluster of differentiation 40 (CD40) is a costimulatory receptor that is expressed by antigen- presenting cells (APCs), B cells, and tumor cells, and plays an important role in innate and adaptive immunity. CD40 interacts with its ligand, CD40L, which is found primarily on activated CD4+ T cells. After its ligation, CD40 is activated and then binds to TNF receptor associated factors (TRAFs), which functions via a wide range of downstream signaling pathways, resulting in the subsequent production of surface and secreted molecules that ultimately impact both humoral and cellular immunity, and can eventually lead to inflammatory responses. Engagement by CD40L expressed on T cells with CD40 expressed on dendritic cells (DCs) induces positive signaling that leads to expression of CD80 / 86 and the production of signaling molecules that skews towards Th1 differentiation in CD4+ T cells. In addition, CD40L signaling in T cells induces IFN-y production. DCs activated via the CD40 / CD40L axis also mediate crosstalk among multiple lymphocytes to regulate, for example, B cell class switching or regulatory T cell activation.

[0396] CD40 plays an important role in the innate and adaptive immune response, and dysregulation of CD40 has been implicated in a number of disease states. In embodiments, exemplary diseases treated by anti-CD40 agonists herein include autoimmune diseases, neurodegenerative diseases, and cancers, For example, in non-limiting embodiments, methods of treatment herein include addressing one or more symptoms of autoimmune thyroiditis, type 1 diabetes, inflammatory bowel disease (IBD), psoriasis, multiple sclerosis (MS), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE), various cancers, traumatic brain injury (TBI), Alzheimer’s Disease (AD), Parkinson’s Disease (PD), stroke, epilepsy, nerve injury, Amyotrophic lateral sclerosis (ALS), and myasthenia gravis, for example, as described in Peters et al., Semin Immunol. 2009. 21 (5):293-300; and Ots et al, Int J Mol Sci. 2022. 23(8): 4115. Use of CD40 modulators as a therapeutic modality has been studied extensively, including for treatment of tumors (Li and Wang, Oncol. Lett. 20: 176, 2020; and Bullock, Cell and Mol Immunol. 2022. (19)14-22). Currently, use of therapeutic CD40 agonists is hindered by the occurrence of adverse events, such as storms of cytokine release, hepatotoxicity, and thromboembolic events, which has been observed in patients treated with anti-CD40 antibodies. Furthermore, dose-dependent T-cell margination after CD40 agonist administration to patients has been observed, resultingPage 1OO of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 in counter-productive immune-dampening effects of the treatment. Intratumoral delivery of CD40 moieties lessens but does not abrogate the above adverse effects.

[0397] In aspects, described herein are methods of treating autoimmune disease using pharmaceutical compositions of the agonists described herein. In embodiments, the method of treatment includes administration to target / treat one or more immune cells expressing CD40 and / or its ligand (CD40L). In embodiments, the method of treatment includes using an anti-CD40 agonist that blocks endogenous CD40L. In embodiments, agonists herein target CD40-CD40L signaling axis (and other signaling pathways) as a novel therapeutic option for autoimmune disorders.

[0398] CD40-CD40L signaling plays a central role in co-stimulation and regulation of the immune response via activation of immune cells expressing CD40. Imbalance of the CD40-CD40L co-stimulatory pathway has been reported in many autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjogren's syndrome, autoimmune nephritis, inflammatory bowel disease (IBD), type 1 diabetes (T1 D), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), autoimmune skin diseases, allograft and transplant rejection, graft versus host disease (GVHD), Graves’ disease, myasthenia gravis, among others, thus supporting its role in the breach of immune tolerance that is typical of these diseases. In embodiments, the autoimmune disease treated by agonists herein comprises systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjogren's syndrome, autoimmune nephritis, inflammatory bowel disease (IBD), type 1 diabetes (T1 D), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), autoimmune skin diseases, allograft and transplant rejection, graft versus host disease (GVHD), Graves’ disease, and myasthenia gravis.

[0399] Several anti-CD40L antibodies have been studied in human clinical trials, such as ruplizumab (lgG1), toralizumab (lgG1), dapirolizumab pegol (Fab’), letolizumab (Fc-modified lgG1), and VIB4920 (Tn3 fusion), for example, as described in Karnell et al., “Targeting the CD40-CD40L pathway in autoimmune diseases: Humoral immunity and beyond,” Adv Drug Deliv Rev. (2019), Vol. 141 , pp: 92-103, the entire contents are incorporated by reference herein. Several anti-CD40 receptor antibodies have been studied in human clinical trials, such as CFS533 (Fc-modified lgG1), bleselumab (lgG4), BI-655064 (humanized lgG1), cd5D12 (lgG4), and FFP104 (lgG4), for example, as described in Karnell et al., 2019. However, early trials revealed grave safety complications associated with targeting the CD40-CD40L signaling axis.

[0400] Without wishing to be bound by theory, the high concentration of solvent-exposed phosphorylation on the agonists herein improve one of more aspects of its use for treating cancer and / or autoimmune disease Page 101 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005(e. g . , modulates retention within a tumor microenvironment, release to draining lymph nodes, abscopal effects, systemic administered due to lack of metal hydroxide and / or metal complexing, dampening / reduction of toxicity associated with targeting CD40 / CD40L signaling axis, etc.), in comparison to a cognate molecule (e.g., anti- CD40 antibody) that lacks the phosphorylation. Without wishing to be bound by theory, the improved intratumoral and abscopal effects observed in cancer models relates to potential organospecific and systemic benefits in treating autoimmune disease. Additionally, and without wishing to be bound by theory, the anti- CD40 agonists herein exhibit these effects without the need for adsorption to a metal hydroxide through the metal hydroxide-binding domain. In embodiments, the anti-CD40 agonists herein exhibit improved in vivo effects in comparison to a cognate anti-CD40 agonists complexed with metal hydroxide (e.g., aluminum hydroxide), and / or exhibit improved in vivo effects in comparison to a cognate anti-CD40 binding agent (e.g., antibody) lacking the metal hydroxide polypeptide.

[0401] In embodiments, the agonist is phosphorylated at one or more phosphorylation sites in a metal hydroxide-binding polypeptide portion of the molecule. In embodiments, the phosphorylation (without metal or metal hydroxide anchoring) permits routes of administration that metal or metal hydroxide anchoring preclude, such as intravenous administration. Additionally, in embodiments, phosphorylation without metal or metal hydroxide anchoring functions, in part, to address toxicities associated with anti-CD40 antibodies, likely mediated via an exposed Fc domain.

[0402] In embodiments, the pharmaceutical composition that is administered to a subject in need thereof is formulated for paternal administration. In embodiments, the pharmaceutical composition administered by intratumoral injection or peritumoral injection. In embodiments, the CD40 agonist is not part of a metal hydroxide complex, and is suitable to be formulated for non-localized paternal administration, such as intravenous injection. Without wishing to be bound by theory, metal hydroxide-complexed proteins are not likely to be tolerated via systemic administration due to the potential immunogenic (e.g., adjuvancy) effects of metal hydroxides, such as alum, and / or may be hindered in their tissue penetrance and / or half-life (e.g., may be cleared faster from blood) due to the presence of the metal hydroxide particles.

[0403] In embodiments, the agonist is administered to one or more tumor-draining lymph nodes, or is suitable to traverse to one or more tumor-draining lymph nodes.

[0404] In embodiments, the cancer is a carcinoma, sarcoma, myeloma, leukemia, lymphoma, mixed type cancer, or a metastatic cancer. In embodiments, the cancer is or comprises a solid tumor.Page 102 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0405] In embodiments, the agonist is administered at a dose of about or at least about 0.05 mg / kg to 5 mg / kg, including doses therebetween.

[0406] In some embodiments, a method of treating a subject with a tumor comprises a step of: treating a subject with a complex comprising: a fusion polypeptide comprising an immunomodulatory polypeptide that comprises an immune agonist moiety and a metal-hydroxide binding polypeptide and a metal hydroxide. In some embodiments, a method of treating a subject with a tumor comprises administering a fusion polypeptide comprising: an immunomodulatory polypeptide that comprises an immune agonist moiety and a metal- hydroxide binding polypeptide, wherein a fusion polypeptide is formulated with a metal hydroxide.

[0407] In some embodiments, a complex is administered as a monotherapy. In some embodiments, a complex is administered in combination with a second therapeutic. In some embodiments, a complex is administered to a subject wherein a subject has received or is receiving therapy with at least one additional therapeutic.

[0408] Fusion polypeptides and / or preparations and / or complexes thereof of the present disclosure, among otherthings, are useful for treating a subject with atumor. Non-limiting examples of diseases associated with a tumor include cancer (e.g., carcinoma, sarcoma, metastatic diseases or hematopoietic neoplastic disorders). A tumor, including a metastatic tumor, can arise from a plurality of primary tumor types. For example, and without limitation, in some embodiments, a tumor or metastatic tumor can arise from a primary tumor of the kidney (e.g., renal cell carcinoma), head and neck (e.g., head and neck squamous cell carcinoma), prostate, breast (e.g., triple-negative), colon, skin (e.g., melanoma, Merkel cell carcinoma, cutaneous T-cell lymphoma, cutaneous squamous cell carcinoma, basal cell carcinoma), lung (e.g., non-small cell lung cancer), and pancreas. Accordingly, fusion polypeptides and preparations thereof disclosed herein, including fusion polypeptide metal-hydroxide complexes and preparations thereof, can be administered to subject who has cancer.

[0409] It will be appreciated by those skilled in the art that amounts of a fusion polypeptide-metal hydroxide complex, fusion polypeptide or a preparation thereof sufficient to reduce tumor growth and size, or a therapeutically effective amount, will vary not only on the particular compounds or preparations selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the patient's physician or pharmacist and / or based upon clinical guidelines. The length of time during which the compounds used in the instant method will be given varies on an individual basis and / or be based upon clinical guidelines.Page 103 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0410] In some embodiments, a method of treating a subject with a tumor (e.g, cancer) comprises a step of treating the subject with a complex comprising a fusion polypeptide comprising an immunomodulatory polypeptide that comprises an immune agonist moiety and a metal-hydroxide binding polypeptide and a metal hydroxide. In some embodiments, a fusion polypeptide and a metal-hydroxide are formulated together. Formulated together, for example, comprises a pre-formed complex of fusion polypeptide and metal-hydroxide. In some embodiments, a fusion polypeptide and metal-hydroxide are mixed immediately prior to administration.

[0411] In some embodiments, a method of treating a subject with a tumor (e.g., cancer) comprises treating a subject with a complex wherein a complex is administered by intratumoral injection. In some embodiments, a method of treating a subject with a tumor (e.g., cancer) comprises treating a subject with a complex wherein a complex is administered by peritumoral injection. In some embodiments, a method of treating a subject with a tumor (e.g., cancer) comprises treating a subject with a complex wherein a complex is administered to a tumor-draining lymph node or lymph nodes.

[0412] Methods taught by the present invention can involve administration of a therapeutically effective amount of a particular agent. A therapeutically effective amount is an amount sufficient to achieve (in principle, for a subject of comparable characteristics, such as species, body type, size, extent of disease or disorder, degree or type of symptoms, history of responsiveness, and / or overall health) an intended biological or medical response or therapeutic benefit in a tissue, system or subject. For example, a desirable response may include one or more of: delaying or preventing the onset of a medical condition, disease or disorder, slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the condition, bringing about ameliorations of the symptoms of the condition, and curing the condition. In some embodiments, a therapeutically effective amount is an amount sufficient to achieve an intended biological or medical response is characterized by the potency of signaling (e.g, receptor signaling) induced by the particular agent.

[0413] In certain embodiments, a therapeutically effective amount is an amount sufficient to achieve an intended biological or medical response is characterized by reduction in tumor size and growth, ablation of a tumor, reduction of metastatic growth, rate and / or spread, tumor cell senescence, tumor cell death, upregulation of innate immunomodulatory molecules, enhanced immune response (e.g. increased antigen presentation by DCs, increased T-cell activation, proliferation and / or infiltration, increased macrophage differentiation and / or activation, etc.), increased overall survival, increased progression-free survival, or a combination thereof.Page 104 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005

[0414] When combinations of therapeutic agents are administered, the amount of any individual agent required in the combination may be different from the amount required of that same agent to achieve its therapeutic effect alone. In some cases, synergies between or among therapeutic agents used in a combination may reduce amounts required; in other cases, inhibitory interactions may increase amounts required. Thus, in general, therapeutically effective amounts of a combination of agents may utilize different absolute amounts of the agents than constitute therapeutically effective amounts of the agents individually.Combination therapies

[0415] In some embodiments, fusion polypeptide metal-hydroxide complexes or preparations thereof can be administered in combination with a second therapeutic agent. A second therapeutic agent may be selected from a variety of available anti-tumor agents known in the art. In some embodiments, a second therapeutic agent is administered prior to administration of a fusion polypeptide metal-hydroxide complex. In some embodiments, a second therapeutic agent is administered concurrently with a fusion polypeptide metal- hydroxide complex. In some embodiments, a second therapeutic agent is administered after administration with a fusion polypeptide metal-hydroxide complex.

[0416] For example, in some embodiments, a second therapeutic is radiation (e.g., ionizing radiation). In some embodiments, an amount of ionizing radiation administered is between about 1 Gy and about 1 ,000 Gy, about 5 Gy and about 900 Gy, about 10 Gy to about 800 Gy, about 10 Gy to about 700 Gy, about 10 Gy to about 600 Gy, about 10 Gy to about 500 Gy, about 10 Gy to about 400 Gy, about 10 Gy to about 300 Gy, about 10 Gy to about 200 Gy, about 10 Gy to about 100 Gy, about 5 Gy and about 15 Gy, between about 7.5 Gy and about 12 Gy, or between about 10 Gy and about 12 Gy. In some embodiments, an amount of ionizing radiation administered is about 12 Gy. In some embodiments, an amount of ionizing radiation is greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1 ,000 Gy. In some embodiments, an amount of ionizing radiation is less than about 1 ,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50 Gy.

[0417] For example, in some embodiments, a second therapeutic agent is a chemotherapeutic agent. In some embodiments, a chemotherapeutic agent may be a targeted therapy (e.g., BRAF inhibitor, MEK inhibitor, etc.). In some embodiments, a chemotherapeutic agent may be any approved chemotherapeutic agent. For example, and without limitation, a chemotherapeutic agent can be one or more of adriamycin, anastrozole, cyclophosphamide,...

Claims

Attorney Docket: AN K-005PC / 138388-5005CLAIMSWhat is claimed is:1 . A CD40 agonist comprising: an antibody or antibody-format molecule that binds CD40; and a metal hydroxide-binding polypeptide comprising an amino acid sequence having one or more phosphorylation sites.

2. The CD40 agonist of claim 1 , wherein the CD40 agonist is substantially lacking a metal hydroxide or metal and / or is substantially not complexed with a metal hydroxide or metal.

3. The CD40 agonist of claim 1 or 2, wherein the metal hydroxide-binding polypeptide is fused to a first chain of the antibody or antibody-format molecule, optionally wherein the first chain comprises a heavy chain or a heavy variable domain, and optionally fused at the C-terminus.

4. The CD40 agonist of any one of claims 1-3, wherein the antibody or antibody-format molecule comprises a Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv (scFv), diabody, single domain antibody (sdAb), single domain heavy chain antibody (VHH, VNAR), linear antibody, monoclonal antibody, polyclonal antibody, bispecific antibody, multi-specific antibody, chimeric antibody, humanized antibody, human antibody, non-human antibody, or a fusion protein comprising an antigen-binding portion of an antibody.

5. The CD40 agonist of claim 4, wherein the antibody or antibody-format molecule comprises one or more Fc domain.

6. The CD40 agonist of any one of claims 1-5, wherein the antibody or antibody-format molecule is isotype selected from IgA, IgG, IgE, or IgM, optionally selected from lgG1, lgG2, lgG3, and lgG4, and further optionally lgG2.Page 233 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-50057. The CD40 agonist of any one of claims 1-6, wherein the CD40 agonist comprises a full-length immunoglobulin comprising two heavy chains and two light chains, wherein the metal hydroxide- binding polypeptide is fused to the C-terminus of one or both heavy chains.

8. The CD40 agonist of any one of claims 1-7, wherein the antibody or antibody-format molecule comprises:(i) 1 , 2, or 3 complementarity determining regions (CDRs) having an amino acid sequence having 1,2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 1 , 2, and 3, or 1, 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 1, 2, and 3; and1 , 2, or 3 CDRs having an amino acid sequence having 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NO: 6, FTS, and SEQ ID NO: 8, or 1, 2, or 3 CDRs having the amino acid sequence of SEQ ID NO: 6, FTS, or SEQ ID NO: 8; optionally comprising the CDRs of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, FTS, and SEQ ID NO: 8;(ii) 1 , 2, or 3 CDRs having an amino acid sequence having 1, 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 11 , 12, and 13, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 11 , 12, and 13; and1 , 2, or 3 CDRs having an amino acid sequence having 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 16, 17, and 18, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 16, 17, and 18; optionally comprising the CDRs of SEQ ID NOs: 11 , 12, 13, 16, 17, and 18;(iii) 1 , 2, or 3 CDRs having an amino acid sequence having 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 21 , 22, and 23, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 21, 22, and 23; and1 , 2, or 3 CDRs having an amino acid sequence having 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 26, 27, and 28, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 26, 27, and 28;Page 234 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 optionally comprising the CDRs of SEQ ID NOs: 21, 22, 23, 26, 27, and 28;(iv) 1, 2, or 3 CDRs having an amino acid sequence having 1, 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 31, 32, and 33, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 31 , 32, and 33; and1, 2, or 3 CDRs having an amino acid sequence having 1, 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 36, 37, and 38, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 36, 37, and 38; optionally comprising the CDRs of SEQ ID NOs: 31, 32, 33, 36, 37, and 38;(v) 1, 2, or 3 CDRs having an amino acid sequence having 1, 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 41, 42, and 43, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 41 , 42, and 43; and1, 2, or 3 CDRs having an amino acid sequence having 1, 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 46, 47, and 48, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 46, 47, and 48; optionally comprising the CDRs of SEQ ID NOs: 41, 42, 43, 46, 47, and 48;(vi) 1, 2, or 3 CDRs having an amino acid sequence having 1, 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NO: 51 , SEQ ID NO: 52, and LDY, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NO: 51 , SEQ ID NO: 52, and LDY; and1, 2, or 3 CDRs having an amino acid sequence having 1, 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 56, 57, and 58, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 56, 57, and 58; optionally comprising the CDRs of SEQ ID NO: 51, SEQ ID NO: 52, LDY, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58;(vii) 1, 2, or 3 CDRs having an amino acid sequence having 1, 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 61, 62, and 63, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 61 , 62, and 63; andPage 235 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-50051 , 2, or 3 CDRs having an amino acid sequence having 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 66, 67, and 68, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 66, 67, and 68; optionally comprising the CDRs of SEQ ID NOs: 61 , 62, 63, 66, 67, and 68;(viii) 1 , 2, or 3 CDRs having an amino acid sequence having 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 71 , 72, and 73, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 71 , 72, and 73; and1 , 2, or 3 CDRs having an amino acid sequence having 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 76, 77, and 78, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 76, 77, and 78; optionally comprising the CDRs of SEQ ID NOs: 71 , 72, 73, 76, 77, and 78;(ix) 1 , 2, or 3 CDRs having an amino acid sequence having 1, 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 81 , 82, and 83, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 81 , 82, and 83; and1 , 2, or 3 CDRs having an amino acid sequence having 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 86, 87, and 88, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 86, 87, and 88; optionally comprising the CDRs of SEQ ID NOs: 81 , 82, 83, 86, 87, and 88;(x) 1, 2, or 3 CDRs having an amino acid sequence having 1, 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 91 , 92, and 93, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 91 , 92, and 93; and1 , 2, or 3 CDRs having an amino acid sequence having 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 96, 97, and 98, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 96, 97, and 98; optionally comprising the CDRs of SEQ ID NOs: 91 , 92, 93, 96, 97, and 98; or(xi) 1 , 2, or 3 CDRs having an amino acid sequence having 1, 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 101 , 102,Page 236 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 and 103, or 1, 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 101 , 102, and / or 103; and1 , 2, or 3 CDRs having an amino acid sequence having 1 , 2, or 3 mutations selected from substitutions or deletions with respect to the amino acid sequence of SEQ ID NOs: 106, 107, and 108, or 1 , 2, or 3 CDRs having the amino acid sequence of SEQ ID NOs: 106, 107, and 108; optionally comprising the CDRs of SEQ ID NOs: 101 , 102, 103, 106, 107, and 108.

9. The CD40 agonist of claim 8, wherein the antibody or antibody-format molecule comprises:(i) the CDRs of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, FTS, and SEQ ID NO:8, and a variable heavy domain (VH) having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 4, or having the amino acid sequence of SEQ ID NO: 4; and / or a variable light domain (VL) about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 9, or having the amino acid sequence of SEQ ID NO: 9; optionally comprising a VH having the amino acid sequence of SEQ ID NO: 4 and a VL having the amino acid sequence of SEQ ID NO: 9;(ii) the CDRs of SEQ I D NOs: 11 , 12, 13, 16, 17, and 18, and a VH having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 14, or having the amino acid sequence of SEQ ID NO: 14; and / or a VL about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or atPage 237 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 19, or having the amino acid sequence of SEQ ID NO: 19; optionally comprising a VH having the amino acid sequence of SEQ ID NO: 14 and a VL having the amino acid sequence of SEQ ID NO: 19;(iii) the CDRs of SEQ ID NOs: 21 , 22, 23, 26, 27, and 28, and a VH having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 24, or having the amino acid sequence of SEQ ID NO: 24; and / or a VL about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 29, or having the amino acid sequence of SEQ ID NO: 29; optionally comprising a VH having the amino acid sequence of SEQ ID NO: 24 and a VL having the amino acid sequence of SEQ ID NO: 29;(iv) the CDRs of SEQ ID NOs: 31 , 32, 33, 36, 37, and 38, and a VH having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 34, or having the amino acid sequence of SEQ ID NO: 34; and / or a VL about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 39, or having the amino acid sequence of SEQ ID NO: 39; optionally comprising a VH having the amino acid sequence of SEQ ID NO: 34 and a VL having the amino acid sequence of SEQ ID NO: 39;(v) the CDRs of SEQ ID NOs: 41, 42, 43, 46, 47, and 48, and a VH having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 44, or having the amino acid sequence of SEQ ID NO: 44; and / or Page 238 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 a VL about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 49, or having the amino acid sequence of SEQ ID NO: 49; optionally comprising a VH having the amino acid sequence of SEQ ID NO: 44 and a VL having the amino acid sequence of SEQ ID NO: 49;(vi) the CDRs of SEQ ID NO: 51, SEQ ID NO: 52, LDY, SEQ ID NO: 56, SEQ ID NO: 57, and SEQID NO: 58, and a VH having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 54, or having the amino acid sequence of SEQ ID NO: 54; and / or a VL about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 59, or having the amino acid sequence of SEQ ID NO: 59; optionally comprising a VH having the amino acid sequence of SEQ ID NO: 54 and a VL having the amino acid sequence of SEQ ID NO: 59;(vii) the CDRs of SEQ ID NOs: 61 , 62, 63, 66, 67, and 68, and a VH having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 64, or having the amino acid sequence of SEQ ID NO: 64; and / or a VL about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 69, or having the amino acid sequence of SEQ ID NO: 69; optionally comprising a VH having the amino acid sequence of SEQ ID NO: 64 and a VL having the amino acid sequence of SEQ ID NO: 69;(viii) the CDRs of SEQ ID NOs: 71, 72, 73, 76, 77, and 78, andPage 239 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 a VH having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 74, or having the amino acid sequence of SEQ ID NO: 74; and / or a VL about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 79, or having the amino acid sequence of SEQ ID NO: 79; optionally comprising a VH having the amino acid sequence of SEQ ID NO: 74 and a VL having the amino acid sequence of SEQ ID NO: 79;(ix) the CDRs of SEQ ID NOs: 81, 82, 83, 86, 87, and 88, and a VH having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 84, or having the amino acid sequence of SEQ ID NO: 84; and / or a VL about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 89, or having the amino acid sequence of SEQ ID NO: 89; optionally comprising a VH having the amino acid sequence of SEQ ID NO: 84 and a VL having the amino acid sequence of SEQ ID NO: 89;(x) the CDRs of SEQ ID NOs: 91, 92, 93, 96, 97, and 98, and a VH having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 94, or having the amino acid sequence of SEQ ID NO: 94; and / or a VL about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 99, or having the amino acid sequence of SEQ ID NO: 99;Page 240 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 optionally comprising a VH having the amino acid sequence of SEQ ID NO: 94 and a VL having the amino acid sequence of SEQ ID NO: 99; or(xi) the CDRs of SEQ ID NOs: 101 , 102, 103, 106, 107, and 108, and a VH having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 104, or having the amino acid sequence of SEQ ID NO: 104; and / or a VL about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 109, or having the amino acid sequence of SEQ ID NO: 109; optionally comprising a VH having the amino acid sequence of SEQ ID NO: 104 and a VL having the amino acid sequence of SEQ ID NO: 109.

10. The CD40 agonist of claim 9, wherein the antibody or antibody-format molecule comprises:(i) the CDRs of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, FTS, and SEQ ID NO:8, and a heavy chain (HC) having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 5, or having the amino acid sequence of SEQ ID NO: 5; and / or a light chain (LC) about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 10, or having the amino acid sequence of SEQ ID NO: 10; optionally comprising a HC having the amino acid sequence of SEQ ID NO: 5 and a LC having the amino acid sequence of SEQ ID NO: 10;(ii) the CDRs of SEQ I D NOs: 11 , 12, 13, 16, 17, and 18, andPage 241 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 a HC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 15, or having the amino acid sequence of SEQ ID NO: 15; and / or a LC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 20, or having the amino acid sequence of SEQ ID NO: 20; optionally comprising a HC having the amino acid sequence of SEQ ID NO: 15 and a LC having the amino acid sequence of SEQ ID NO: 20;(iii) the CDRs of SEQ ID NOs: 21, 22, 23, 26, 27, and 28, and a HC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 25, or having the amino acid sequence of SEQ ID NO: 25; and / or a LC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 30, or having the amino acid sequence of SEQ ID NO: 30; optionally comprising a HC having the amino acid sequence of SEQ ID NO: 25 and a LC having the amino acid sequence of SEQ ID NO: 30;(iv) the CDRs of SEQ ID NOs: 31, 32, 33, 36, 37, and 38, and a HC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 35, or having the amino acid sequence of SEQ ID NO: 35; and / or a LC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 40, or having the amino acid sequence of SEQ ID NO: 40;Page 242 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 optionally comprising a HC having the amino acid sequence of SEQ ID NO: 35 and a LC having the amino acid sequence of SEQ ID NO: 40;(v) the CDRs of SEQ ID NOs: 41 , 42, 43, 46, 47, and 48, and a HC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 45, or having the amino acid sequence of SEQ ID NO: 45; and / or a LC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 50, or having the amino acid sequence of SEQ ID NO: 50; optionally comprising a HC having the amino acid sequence of SEQ ID NO: 45 and a LC having the amino acid sequence of SEQ ID NO: 50;(vi) the CDRs of SEQ ID NO: 51, SEQ ID NO: 52, LDY, SEQ ID NO: 56, SEQ ID NO: 57, and SEQID NO: 58, and a HC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 55, or having the amino acid sequence of SEQ ID NO: 55; and / or a LC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 60, or having the amino acid sequence of SEQ ID NO: 60; optionally comprising a HC having the amino acid sequence of SEQ ID NO: 55 and a LC having the amino acid sequence of SEQ ID NO: 60;(vii) the CDRs of SEQ ID NOs: 61 , 62, 63, 66, 67, and 68, and a HC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 65, or having the amino acid sequence of SEQ ID NO: 65; and / orPage 243 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 a LC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 70, or having the amino acid sequence of SEQ ID NO: 70; optionally comprising a HC having the amino acid sequence of SEQ ID NO: 65 and a LC having the amino acid sequence of SEQ ID NO: 70;(viii) the CDRs of SEQ ID NOs: 71, 72, 73, 76, 77, and 78, and a HC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 75, or having the amino acid sequence of SEQ ID NO: 75; and / or a LC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 80, or having the amino acid sequence of SEQ ID NO: 80; optionally comprising a HC having the amino acid sequence of SEQ ID NO: 75 and a LC having the amino acid sequence of SEQ ID NO: 80;(ix) the CDRs of SEQ ID NOs: 81 , 82, 83, 86, 87, and 88, and a HC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 85, or having the amino acid sequence of SEQ ID NO: 85; and / or a LC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 90, or having the amino acid sequence of SEQ ID NO: 90; optionally comprising a HC having the amino acid sequence of SEQ ID NO: 85 and a LC having the amino acid sequence of SEQ ID NO: 90;(x) the CDRs of SEQ ID NOs: 91, 92, 93, 96, 97, and 98, and a HC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about Page 244 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 95, or having the amino acid sequence of SEQ ID NO: 95; and / or a LC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 100, or having the amino acid sequence of SEQ ID NO: 100; optionally comprising a HC having the amino acid sequence of SEQ ID NO: 95 and a LC having the amino acid sequence of SEQ ID NO: 100; or(xi) the CDRs of SEQ ID NOs: 101 , 102, 103, 106, 107, and 108, and a HC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 105, or having the amino acid sequence of SEQ ID NO: 105; and / or a LC having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to the amino acid sequence of SEQ ID NOs: 110 or 111 , or having the amino acid sequence of SEQ ID NOs: 110 or 111 ; optionally comprising a HC having the amino acid sequence of SEQ ID NO: 105 and a LC having the amino acid sequence of SEQ ID NO: 110 or 111 .

11. The CD40 agonist of claim 10, wherein the antibody or antibody-format molecule comprises a heavy chain having about or at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 5, and a light chain having about or at least about 95% sequence identity the amino acid sequence of SEQ ID NO: 10.

12. The CD40 agonist of any one of claims 1-11, wherein the antibody or antibody-format molecule comprises one or more mutations that alters, reduces, or ablates binding to an Fc receptor (Fey receptor (FcyR), neonatal Fc receptor (FcRn)), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or antibody-dependent cellular phagocytosis (ADCP), optionally wherein the one orPage 245 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 more mutations is located within a constant portion, heavy chain Fc domain, and / or within a framework region of a variable heavy domain or a variable light domain.

13. The CD40 agonist of any one of claims 1-12, wherein the antibody or antibody-format molecule comprises an Fc domain having one or more mutations at positions selected from 226, 229, 234,235, 237, 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 271 , 272, 279, 280,283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 312, 315, 322, 324,327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 356, 360, 366, 368, 373, 376, 378, 379, 382,388, 389, 394, 398, 405, 407, 414, 416, 419, 430, 434, 435, 437, 438, and 439, or equivalent positions thereto, optionally relative to the lgG1 Fc domain of SEQ ID NO: 190, the lgG2 Fc domain of SEQ ID NO: 192, or the lgG4 Fc domain of SEQ ID NO: 194.

14. The CD40 agonist of claim 13, wherein the Fc domain is an lgG1 isotype and comprises a mutation of one or more residues selected from L234, L235, and P329, relative to SEQ ID NO: 190, or equivalent positions thereto.

15. The CD40 agonist of claim 14, wherein the one or more mutations comprises one or more mutation selected from L234A, L235A, and P329G or P329A, optionally comprising L234A, L235A, and P329A (LALAPA), or L234A, L235A, and P329G (LALAPG).

16. The CD40 agonist of any one of claims 13-15, wherein the Fc domain is an lgG1 isotype and comprises a mutation of one or more residues selected from G237, P238, S267, H268, P271, N297, L328, and A330, relative to SEQ ID NO: 190, or equivalent positions thereto.

17. The CD40 agonist of claim 16, wherein the one or more mutations comprise G237D, P238D, S267E, H268D, P271 G, N297A, L328F, A330R, optionally selected from N297A, S267E, S627E / L328F, G237D / P238D / P271G / A330R, and G237D / P238D / H268D / P271 G / A330R.

18. The CD40 agonist of claim 13, wherein the Fc domain is an I gG2 isotype and comprises a mutation of one or more residues selected from C127 and C232, relative to SEQ ID NO: 192, or equivalent positions thereto, optionally selected from C127S and C232S.Page 246 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-500519. The CD40 agonist of any one of claims 1-18, wherein the metal hydroxide-binding polypeptide comprises about or at least about 1 phosphorylated amino acid to about or at least about 10 phosphorylated amino acids, optionally wherein the one or more of the phosphorylated amino acids is selected from phosphoserine, phoshotyrosine, and phosphothreonine, further optionally wherein the one or more of the phosphorylated amino acids is phosphoserine.

20. The CD40 agonist of any one of claims 1-19, wherein the one or more kinase target motifs comprises an amino acid consensus sequence selected from S-X-E (Fam20C), S-X-pS(Fam20C), S-X-Q-X-X- D-E-E (SEQ ID NO: 364, Fam20C), S-X-Q-X-X-D-E (SEQ ID NO: 365, Fam20C), R-R-X-S / T-Cp (protein kinase A (PKA)), R-R / K-X-S-cp (cAMP-dependent protein kinase), S / T-P-X-K / R (cyclin- dependent kinase (CDK), P-X-S / T-P (extracellular-regualted kinase-2 (ERK)), DS-X-X-S / T (casein kinase 1 (CK1)), D / E-D / E-D / E-X-X-S / T-cp-pS / pT-X-X-S / T-cp (CK1), S / T-D / E-X-D / E (casein kinase 2 (CK2)), S-X-X-X-pS (glycogen synthase kinase-3 (GSK3)), R-X-X-S / T (calmodulin-dependent protein kinase-2 (CaMK2), l / V / L-Y-X-X-P / F (Abelson murine leukemia virus tyrosine kinase (ABL), E-E-E-Y-F (SEQ ID NO: 366, epidermal growth factor receptor (EGFR)), E-E-l-Y-E / G-X-F (SEQ ID NO: 367, Rous sarcoma virus tyrosine kinase (Src)), Y-M-M-M (SEQ ID NO: 368, insulin receptor tyrosine kinase (IPK)), R-X-R-X-X-S / T (protein kinase B (PKB / AKT)), L / l-X-R-X-X-S / T (protein kinase D (PKD)), R-X-R-X-X-S / T (proviral integration site kinases 1-2 (PIM1-3)), cp-X-R_X-X-S-X-X-X-l / L (AMP-activated protein kinase (AMPK), P / cp-X-S / T-P (mitogen-activated protein kinase), and (p-X- X-S / T (NimA-related kinase), wherein X is any amino acid, cp is a hydrophobic amino acid, pS is a phosphoserine, pT is a phosphothreonine, and a phosphoacceptor amino acid is underlined.

21. The CD40 agonist of claim 20, wherein the metal hydroxide-binding polypeptide comprises an amino acid sequence having one or more phosphorylation sites comprising an amino acid consensus sequence of S-X-E, S-E-pS, or S-X-Q-X-X-D-E (SEQ ID NO: 365), wherein X is any amino acid and pS is a phosphoserine, optionally wherein X is selected from E, S, V, H, Q, and G, further optionally wherein X is E.

22. The CD40 agonist of any one of claims 21, wherein the metal hydroxide-binding polypeptide is or comprises one or more amino acid sequence of GGSEEGG (SEQ ID NO: 371), FQSEEQQ (SEQ IDPage 247 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005NO: 372), orMESEESN (SEQ ID NO: 373), optionally comprising one or more repeats of GGSEEGG (SEQ ID NO: 371).

23. The CD40 agonist of any one of claims 1-22, wherein the metal hydroxide-binding polypeptide comprises one or more spacer sequences comprising about or at least about 1 amino acid to about or at least about 20 amino acids, optionally wherein the one or more spacer sequences separate two or more adjacent phosphorylation sites.

24. The CD40 agonist of claim 23, wherein the one or more spacer sequences comprises glycine, serine, glutamic acid, and / or cysteine residues, optionally wherein the one or more spacer sequences comprises about or at least about 4 glycine residues.

25. The CD40 agonist of claim 24, wherein the metal hydroxide-binding polypeptide comprises two or more S-X-E phosphorylation sites, and wherein the two or more S-X-E phosphorylation sites are separated by a spacer sequence comprising 4 glycine residues.

26. The CD40 agonist of any one of claims 1-25, wherein the metal hydroxide-binding polypeptide comprises about or at least about 3 amino acids to about or at least about 100 amino acids, or any length therebetween.

27. The CD40 agonist of any one of claims 1-26, wherein the metal hydroxide-binding polypeptide comprises about or at least about 1 phosphorylation site to about or at least about 10 phosphorylation site, optionally comprising about or at least about 2 phosphorylation sites to about or at least about 5 phosphorylation sites, and / or wherein the metal hydroxide-binding polypeptide comprises about or at least about 1 phosphorylated residue to about or at least about 10 phosphorylated residues, optionally comprising about or at least about 2 phosphorylated residues to about or at least about 5 phosphorylated residues.

28. The CD40 agonist of any one of claims 1-27, wherein the metal hydroxide-binding polypeptide comprises an amino acid sequence having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or atPage 248 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 128-143, 371-396, and 399-401.

29. The CD40 agonist of claim 28, wherein the metal hydroxide-binding polypeptide is or comprises an amino acid sequence of any one of SEQ ID NOs: 128-143, 371-396, and 399-401.

30. The CD40 agonist of any one of claims 1-29, further comprising an amino acid linker between the antibody or antibody-format molecule and the metal hydroxide-binding polypeptide.

31. The CD40 agonist of claim 30, wherein the amino acid linker comprises about or at least about 1 amino acid to about or at least about 50 amino acids, including lengths therebetween, optionally wherein the amino acid linker comprises glycine, serine, glutamic acid, and / or cysteine residues, optionally comprising glycine and serine residues or glycine and glutamic acid residues.

32. The CD40 agonist of claim 30 or 31 , wherein the amino acid linker is or comprises one or more sequences of G, S, GG, SS, GS, SG, GGS, SEG, SSG, GGGG (SEQ ID NO: 402), SGGG (SEQ ID NO: 415), GGGS (SEQ ID NO: 421), GGGGS (SEQ ID NO: 214), GGGGEGGGG (SEQ ID NO: 416), GGGGSGGGG (SEQ ID NO: 417), GGGGSGGGGS (SEQ ID NO: 215), and (GlyxSer)y, wherein x and y is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, and 10.

33. The CD40 agonist of claim 1 , comprising:(i) an lgG2 antibody configured to bind human CD40 comprising: complementarity determining regions (CDRs) of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, FTS, and SEQ ID NO: 8; a variable heavy domain (VH) having about or at least about 90%, sequence identity to the amino acid sequence of SEQ ID NO: 4; and a variable light domain (VL) having about or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 9; and(ii) a metal hydroxide-binding polypeptide comprising:Page 249 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 an amino acid sequence having one or more phosphorylation sites and fused to the C- terminus of a heavy chain of the I gG2 antibody, optionally fused via an amino acid linker sequence, and wherein the metal hydroxide-binding polypeptide comprises at least about 2 phosphorylation sites, each comprising the sequence S-E-E.

34. The CD40 agonist of claim 33, wherein the I gG2 antibody comprises: a heavy chain (HC) having about or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, optionally comprising a heavy chain having the amino acid sequence of SEQ ID NO: 5; and a light chain (LC) having about or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 10, optionally comprising a light chain having the amino acid sequence of SEQ ID NO: 10.

35. The CD40 agonist of claim 33 or 34, wherein the metal hydroxide-binding polypeptide comprises one or more amino acid sequences of GGSEEGG (SEQ ID NO: 371), FQSEEQQ (SEQ ID NO: 372), or MESEESN (SEQ ID NO: 373), optionally comprising one or more repeats of GGSEEGG (SEQ ID NO: 371).

36. The CD40 agonist of any one of claims 33-35, wherein the metal hydroxide-binding polypeptide comprises about or at least about 3 amino acids to about or at least about 100 amino acids, or any length therebetween.

37. The CD40 agonist of any one of claims 33-36, wherein the metal hydroxide-binding polypeptide comprises an amino acid sequence having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, or about or at least about 99% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 128-143, optionally wherein the metal hydroxide- binding polypeptide is or comprises the amino acid sequence of any one of SEQ ID NOs: 128-143.

38. A complex comprising the CD40 agonist of any one of claims 1-37 and a metal hydroxide.Page 250 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-500539. The complex of claim 38, wherein the metal hydroxide is aluminum hydroxide.

40. A nucleic acid molecule comprising one or more nucleic acid sequences encoding the CD40 agonist of any one of claims 1-37.

41. The nucleic acid molecule of claim 40, wherein the one or more nucleic acid sequences comprises: a first nucleic acid sequence encoding a light chain of an anti-CD40 antibody; and a second nucleic acid sequence encoding a heavy chain of an anti-CD40 antibody, and optionally wherein the heavy chain is fused to a metal hydroxide-binding polypeptide at the C-terminus.

42. The nucleic acid molecule of claim 40 or 41 , wherein the nucleic acid molecule comprises a sequence that facilitates cellular export of the CD40 agonist, optionally an antibody secretion signal.

43. A cell comprising one or more nucleic acid sequences encoding the CD40 agonist of any one of claims 1-37, or the nucleic acid molecule of any one of claims 40-42, optionally wherein the cell is suitable to express the CD40 agonist, and optionally wherein the cell comprises one or more nucleic acid sequences encoding a kinase suitable to phosphorylate the CD40 agonist at one or more phosphorylation sites of a metal hydroxide-binding polypeptide, and optionally wherein the kinase is Fam20C.

44. A pharmaceutical composition comprising the CD40 agonist of any one of claims 1-37, or the complex of claim 38 or 39, and one or more pharmaceutically-acceptable excipients, carriers, or diluents.

45. A method of manufacturing a phosphorylated form of the CD40 agonist of any one of claims 1-37, the method comprising co-expressing, in a host cell, the CD40 agonist and a kinase that is suitable for phosphorylating one or more phosphorylation sites in a metal hydroxide-binding polypeptide of the CD40 agonist, optionally wherein the kinase is Fam20C, and optionally wherein the CD40 agonist is encoded on one or more nucleic acid sequences comprising a first nucleic acid sequence encoding a light chain of an anti-CD40 antibody, and a second nucleic acid sequence encoding a heavy chainPage 251 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-5005 of an anti-CD40 antibody, optionally wherein the heavy chain is fused to a metal hydroxide-binding polypeptide at the C-terminus.

46. A method of treating a cancer or an autoimmune disease in a subject in need thereof comprising administering to the subject the pharmaceutical composition of claim 44, wherein the pharmaceutical composition comprises a CD40 agonist, or a complex comprising the CD40 agonist and a metal hydroxide.

47. The method of claim 46, wherein the method comprises administering the complex comprising the CD40 agonist and the metal hydroxide.

48. The method of claim 46 or 47, wherein the metal hydroxide is or comprises aluminum hydroxide.

49. The method of any one of claims 46-48, wherein the pharmaceutical composition comprising the CD40 agonist, and the metal hydroxide, are administered substantially simultaneously, optionally wherein the CD40 agonist and the metal hydroxide are admixed prior to, or substantially simultaneously with, administration.

50. The method of claim 46, wherein the method comprises administering the CD40 agonist without a metal hydroxide.

51. The method of claim 50, wherein the CD40 agonist is not part of a metal hydroxide complex, and is formulated for paternal administration, optionally intravenous injection.

52. The method of any one of claims 46-51, wherein the pharmaceutical composition is formulated for paternal administration, optionally administered by intratumoral injection or peritumoral injection.

53. The method of any one of claims 46-52, wherein the CD40 agonist is administered to one or more tumor-draining lymph nodes, or is suitable to traverse to one or more tumor-draining lymph nodes.Page 252 of 254DBl / 159956110.2Attorney Docket: AN K-005PC / 138388-500554. The method of any one of claims 46-53, wherein the cancer is a carcinoma, sarcoma, myeloma, leukemia, lymphoma, mixed type cancer, or a metastatic cancer, optionally wherein the cancer is or comprises a solid tumor.

55. The method of any one of claims 46-53, wherein the autoimmune disease selected from systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjogren's syndrome, autoimmune nephritis, inflammatory bowel disease (IBD), type 1 diabetes (T1 D), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), autoimmune skin diseases, allograft and transplant rejection, graft versus host disease (GVHD), Graves’ disease, and myasthenia gravis.Page 253 of 254DBl / 159956110.2

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