Compositions and methods of use for binding agents

Abstract

The present disclosure provides a targeted degradation platform technology related to binding agents for degrading endogenous proteins, whether membrane-associated or soluble, using the lysosome pathway for the treatment of various disorders.

Description

COMPOSITIONS AND METHODS OF USE FOR BINDING AGENTSCROSS-REFERENCE[1] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 594,662 filed on October 31, 2023, and U.S. Provisional Patent Application No. 63 / 575,377 filed on April 5, 2024, each of which is incorporated by reference in its entirety.BACKGROUND[2] Targeted protein degradation is a promising new therapeutic strategy compared to conventional inhibition-based therapeutics. Inhibitors rely on sustained, occupancy-driven pharmacology, necessitating high affinity binders capable of abrogating catalytic or binding functions. Inhibiting protein-protein interactions or scaffolding function has been extremely challenging for standard binding-based small molecules. In contrast, protein degraders are catalytic and utilize event-driven pharmacology, alleviating the need for high affinity binders, and durably abrogate all protein functions at once. To date, most degraders are heterobifunctional small molecules that recruit intracellular E3 ubiquitin ligases to a target of interest, which induces ubiquitination of the target protein and its subsequent degradation by the proteasome. As such, degrader technologies such as proteolysis targeting chimeras (PROTACs) have had great success in targeting traditionally challenging proteins. Several PROTACs are currently in clinical trials. Most degrader technologies, including PROTACs, utilize an intracellular mechanism of action and have thus been largely limited to targeting proteins with cytoplasmic domains. However, recent approaches, such as LYTACs have been described for specifically degrading cell surface proteins. These utilize recycling glycan receptors such as the mannose-6-phosphate receptor (M6PR) or asialoglycoprotein receptor (ASGR) to target proteins for internalization and trafficking to the lysosome for degradation. These require complex glycans conjugated to antibodies or to small molecules to achieve degradation of a membrane protein.[3] As a hybrid approach that is broadly applicable to many cell types, we recently described antibody-based PROTACs (AbTACs). AbTACs utilize a standard IgG bispecific antibody format to bring a cell surface E3 ligase (RNF43) into proximity of a membrane protein of interest (POI) to mediate its degradation through the lysosomal pathway. The traditional bispecific IgG scaffold on which the AbTAC is built possesses favorable pharmacokinetic properties relative to LYTACS and other small molecule-based degraders. Furthermore, incontrast to other degradation modalities, such as LYTACS and PROTACS, AbTACs are fully recombinant.[4] Cytokines and growth factors are each a diverse class of soluble extracellular proteins. Upon binding to their cognate receptors on the surface of cells, cytokines and growth factors trigger downstream signaling, leading to internalization of the cytokine-receptor complex. Thus, cytokine-mediated and growth factor-mediated internalization could be co- opted for targeted degradation applications. Cytokine receptor targeting chimeras (KineTACs), which comprise fully recombinant bispecific binding agents, utilize cytokine-mediated internalization of cognate receptors to target relevant cell surface proteins for lysosomal degradation.[5] While targeted protein degradation therapies have shown great promise in treating various diseases, there can be challenges associated with achieving cell specificity. For example, targeted protein degradation therapies may act on several cell types, leading to off- target effects which can result in adverse reactions and interfere with normal cellular functions. This can be particularly problematic when treating cancers and / or autoimmune diseases where treatments for autoimmune diseases which lack cell-type specificity often result in broad immunosuppression, and treatments for cancers which lack cell-type specificity often result in killing many fast-growing cells, not just cancer cells. Consequently, cancer and / or autoimmune disease treatments which lack cell-type specificity can have significant side effects and increase the risk of infections. Thus, improving the selectivity of targeted protein degradation therapies, and developing strategies to target specific cell-types within heterogeneous populations may be crucial for the successful development of targeted protein degradation therapies for the treatment of cancers and / or autoimmune diseases.[6] Autoimmune diseases occur when the immune system mistakenly attacks the body's own tissues, mistaking them for harmful pathogens. This aberrant immune response can lead to various chronic conditions, such as rheumatoid arthritis, lupus, and multiple sclerosis, each characterized by its own set of symptoms and complications. However, managing autoimmune diseases often involves a delicate balance, as suppressing the immune system to control the disease can increase the liability to infections, necessitating careful monitoring and management. In recent years, various regulatory immune proteins and cells have been identified that maintain immune tolerance and prevent autoimmune response. Disruptions in the quality, quantity, and function of these cells have been implicated in autoimmune disease development. Therefore, targeted disruption of these regulatory immune proteins and / or cells could be a promising therapeutic strategy for treating different autoimmune diseases.SUMMARY[7] The present disclosure demonstrates the development of a targeted degradation platform technology, which is comprised of bispecific binding agents that target both therapeutically relevant extracellular proteins, as well as cell surface receptors for receptor- mediated internalization and subsequent lysosomal degradation. The present disclosure also demonstrates methods for regulating intracellular downstream proteins (e.g., K-ras) through targeting cell surface or extracellular upstream proteins (e.g., receptor tyrosine kinases). Regulating intracellular downstream proteins through targeting cell surface or extracellular upstream proteins can drive inhibition and / or activation of signaling pathways (e.g., regulating cell growth, division, and death).[8] Provided herein are methods of degrading an extracellular soluble target protein, the method comprising: contacting the extracellular soluble target protein and a membrane- associated protein with a binding agent, wherein the binding agent comprises: a first binding domain that specifically binds to the extracellular soluble target protein, and a second binding domain that specifically binds to the membrane-associated protein, wherein the membrane- associated protein is associated with a target cell; and wherein the contacting of the extracellular soluble target protein and the membrane-associated protein with the binding agent leads to degradation of the soluble target protein. In some embodiments, the binding agent comprises a bispecific, or a multi-specific binding agent. In some embodiments, the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, bispecific camelid antibody, a bi specific peptibody scFv-Fc, a bispecific IgG, an immunocytokine, an IgG- cytokine, a knob and hole bispecific IgG, a Fc-Fab, a Fc-cytokine, a scFv-Fc-cytokine, a Fab- cytokine, or a knob and hole bispecific Fc-Fab. In some embodiments, the binding agent comprises a third binding domain. In some embodiments, the second binding domain comprises a second binding domain variable heavy chain and a second binding domain variable light chain. In some embodiments, the second binding domain comprises a cytokine, or a biologically active fragment thereof, selected from the group comprising CXCL12, CCL1, CCL2, CCL3, CCL3L1, CCL4, CC4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL11, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, vMIPII, and vCXCl. In some embodiments, the second binding domain specifically binds to one membrane-associated protein. In some embodiments, the membrane-associated protein comprises single-pass and multi-pass membrane proteins. In some embodiments, the membrane-associated protein comprises a tissue-type specific protein. In some embodiments, the membrane-associated protein comprises a cell-type specific protein. In some embodiments, the membrane-associated protein comprises an endogenous internalizing receptor or a membrane-associated ubiquitin E3 ligase. In some embodiments, the endogenous internalizing receptor comprises targeting receptors or recycling receptors. In some embodiments, the membrane-associated protein is selected from one or more of the following: HER2, CD30, CXCR7, EPHA2, CCR8, 0X40, CD79B, Nectin-4, BCMA, integrin 07, IL1RAP, EGFR, CD33, MUC1, ITGB6, CEACAM5, CDH17, CD20, CD22, CD19, BAFF-R, CD38, TROP2, B7-H3, MARCH-1, MARCH-8, MARCH-9, GRAIL, Tissue factor, FOLR1, CD45, c-Kit, and TFRC. In some embodiments, the target cell comprises a microglia cell, endothelial cell, smooth muscle cell, immune cell, plasma cell, B cell, neuronal cell, innate immune cell, lymphocyte, monocyte, granulocyte, T cell, regulatory T cell, effector T cell, activated CD8 T cell, CD8+ T cell, CD4+ T cell, Th 17 cell, macrophage, or dendritic cell. In some embodiments, the membrane-associated protein is enriched on a regulatory T cell when compared to an effector T cell. In some embodiments, the membrane-associated protein is enriched on an effector T cell when compared to a regulatory T cell. In some embodiments, the first binding domain binds to an epitope of the soluble target protein. In some embodiments, the soluble target protein comprises an aggregated protein. In some embodiments, the soluble target protein is selected from one or more of the following: an immune checkpoint protein, an immunomodulatory protein, an inflammatory cytokine, an autoantibody, a shed receptor, and a neuronal aggregate protein. In some embodiments, the soluble target protein is selected from one or more of the following: BAFF, IgG4, TNFa, MICA / B, IL-23, IL-1, IL-2, IL-4, IL-6, IL- 12, IFNy, and A0 protein. In some embodiments, the soluble target protein comprises BAFF. In some embodiments, the soluble target protein comprises IgG4. In some embodiments, the soluble target protein comprises TNFa. In some embodiments, the soluble target protein comprises MICA / B. In some embodiments, the soluble target protein comprises IL-23. In some embodiments, the membrane-associated protein comprises integrin 07. In some embodiments, the target cell comprises a T cell. In some embodiments, the soluble target protein comprises IL-1. In some embodiments, the soluble target protein comprises IL-2. In some embodiments, the soluble target protein comprises IL-4. In some embodiments, the soluble target protein comprises IL-6. In some embodiments, the soluble target protein comprises IL-12. In some embodiments, the soluble target protein comprises IFNy. In some embodiments, the solubletarget protein comprises A0 protein. In some embodiments, the membrane-associated protein is not an Fey receptor. In some embodiments, the A0 protein is an intracellular or extracellular A0 protein. In some embodiments, contacting of the second binding domain to the membrane- associated protein results in the internalization of the membrane-associated protein and the binding agent. In some embodiments, contacting of the first binding domain to the soluble target protein, and the subsequent or simultaneous contacting of the second binding domain to the membrane-associated protein, results in the internalization of the membrane-associated protein, the binding agent, and the soluble target protein, and wherein the soluble target protein is then degraded. In some embodiments, the membrane-associated protein is recycled to the target cell surface following the internalization of the binding agent. In some embodiments, the membrane-bound target protein is an intracellular protein. In some embodiments, the soluble target protein is an intracellular protein. In some embodiments, the method further comprises modulating an intracellular protein comprising targeting an upstream cell surface receptor with a bispecific or a multispecific antibody. In some embodiments, the intracellular protein is a cell signaling protein. In some embodiments, the cell signaling protein is selected from FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lek, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek2, MKK3 / 6, MKK4 / 7, ASK 1, Cot, NIK, Bub, Myt 1, Weel, Casein kinases, PDK1, SGK1, SGK2, SGK3, Aktl, Akt2, Akt3, p90Rsks, p70S6 Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs, Chkl, Chk2, LKB-1, MAPKAPKs, Piml, Pim2, Pim3, IKKs, Cdks, Inks, Erks, IKKs, GSK3a, GSK3P, Cdks, CLKs, PKR, PI3- Kinase, mTor, SAPK / JNK1,2,3, p38s, PKR, DNA-PK, ATM, or ATR. In some embodiments, the cell signaling receptor is selected from H-Ras, K-Ras, or N-Ras. In some embodiments, the modulating of the intracellular protein comprises downregulating, upregulating, or post- translationally modifying the intracellular protein. In some embodiments, the modulating of the intracellular protein comprises modulating the scaffolding of the intracellular protein.[9] Provided herein are methods of degrading a membrane-bound target protein, the method comprising: contacting the membrane-bound target protein and a membrane-associated protein with a binding agent, wherein the binding agent comprises: a first binding domain that specifically binds to the membrane-bound target protein, and a second binding domain that specifically binds to the membrane-associated protein, wherein the membrane-associated protein is associated with a target cell; and wherein the contacting of the membrane-bound target protein and the membrane-associated with the binding agent leads to degradation of themembrane-bound target protein. In some embodiments, the binding agent comprises a bispecific, or a multi-specific binding agent. In some embodiments, the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, an immunocytokine, an IgG-cytokine, a knob and hole bispecific IgG, a Fc-Fab, a Fc-cytokine, a scFv-Fc-cytokine, a Fab-cytokine, or a knob and hole bispecific Fc-Fab. In some embodiments, the second binding domain comprises a second binding domain variable heavy chain and a second binding domain variable light chain. In some embodiments, the second binding domain comprises a cytokine, or a biologically active fragment thereof, selected from the group comprising CXCL12, CCL1, CCL2, CCL3, CCL3L1, CCL4, CC4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL11, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, vMIPII, and vCXCl. In some embodiments, the second binding domain specifically binds to one membrane-associated protein. In some embodiments, the membrane-associated protein comprises single-pass and multi-pass membrane proteins. In some embodiments, the membrane-associated protein comprises a tissue-type specific protein. In some embodiments, the membrane-associated protein comprises a cell-type specific protein. In some embodiments, the membrane-associated protein comprises an endogenous internalizing receptor or a membrane-associated ubiquitin E3 ligase. In some embodiments, the endogenous internalizing receptor comprises targeting receptors or recycling receptors. In some embodiments, the membrane-associated protein is selected from one or more of the following: HER2, CD30, CXCR7, EPHA2, CCR8, 0X40, CD79B, Nectin-4, BCMA, integrin 07, IL1RAP, EGFR, CD33, MUC1, ITGB6, CEACAM5, CDH17, CD20, CD22, CD19, BAFF-R, CD38, TROP2, B7-H3, MARCH-1, MARCH-8, MARCH-9, GRAIL, Tissue factor, FOLR1, CD45, c-Kit, and TFRC. In some embodiments, the target cell comprises a microglia cell, endothelial cell, smooth muscle cell, immune cell, plasma cell, B cell, neuronal cell, innate immune cell, lymphocyte, monocyte, granulocyte, regulatory T cell, T cell, effector T cell, activated CD8 T cell, CD8+ T cell, CD4+ T cell, Th 17 cell, macrophage, or dendritic cell. In some embodiments, the membrane-associated protein is enriched on a regulatory T cell when compared to an effector T cell. In some embodiments, the membrane-associated protein is enriched on an effector T cell when compared to a regulatory T cell. In some embodiments, the first binding domain binds to an epitope of the membrane-bound target protein. In some embodiments, the membrane-bound target protein is selected from one or more of thefollowing: an immune checkpoint protein, an immunomodulatory protein, and an autoantibody. In some embodiments, the membrane-bound target protein comprises a tissue-type specific protein. In some embodiments, the membrane-bound target protein comprises a cell-type specific protein. In some embodiments, the membrane-bound target protein comprises multiple ligand-binding sites. In some embodiments, the membrane-bound target protein is selected from one or more of the following: MerTK, PD-1, ICOS, TIM3, B7H4, RAGE, HLA DQ2.5, GSDMD, TLR7, CD86, MHC-II, MHC-I, ICAM, c-Kit, CD83, CD3i and a4 integrin protein. In some embodiments, the membrane-bound target protein comprises MerTK. In some embodiments, the membrane-bound target protein comprises PD-1. In some embodiments, the membrane-associated protein comprises CD30. In some embodiments, the membrane- associated protein comprises CCR8. In some embodiments, the membrane-associated protein comprises 0X40. In some embodiments, the target cell comprises a regulatory T cell. In some embodiments, the membrane-bound target protein comprises ICOS. In some embodiments, the membrane-associated protein comprises IL1RAP. In some embodiments, the target cell comprises an effector T cell. In some embodiments, the membrane-bound target protein comprises TIM3. In some embodiments, the membrane-bound target protein comprises B7H4. In some embodiments, the membrane-bound target protein comprises RAGE. In some embodiments, the membrane-bound target protein comprises HLA DQ2.5. In some embodiments, the membrane-bound target protein comprises GSDMD. In some embodiments, the membrane-bound target protein comprises TLR7. In some embodiments, the membranebound target protein comprises MHC-II. In some embodiments, the membrane-bound target protein comprises CD86. In some embodiments, the membrane-associated protein comprises MARCH-8. In some embodiments, the membrane-associated protein comprises MARCH-1. In some embodiments, the target cell comprises a T cell. In some embodiments, the membranebound target protein comprises MHC-I. In some embodiments, the membrane-bound target protein comprises ICAM. In some embodiments, the membrane-associated protein comprises MARCH-9. In some embodiments, the target cell comprises a T cell. In some embodiments, the membrane-bound target protein comprises CD83. In some embodiments, the membranebound target protein comprises CD3(^. In some embodiments, the membrane-associated protein comprises GRAIL. In some embodiments, the target cell comprises a T cell. In some embodiments, the membrane-bound target protein comprises a4 integrin. In some embodiments, the membrane-bound target protein is located on the target cell. In some embodiments, the membrane-bound target protein is not located on the surface of a target cell. In some embodiments, the membrane-bound target protein is located on an organelle withinthe target cell. In some embodiments, the membrane-bound target protein is a lysosomal membrane-associated protein. In some embodiments, contacting of the second binding domain to the membrane-associated protein results in the internalization of the membrane-associated protein and the binding agent. In some embodiments, contacting of the first binding domain to the membrane-bound target protein, and the subsequent or simultaneous contacting of the second binding domain to the membrane-associated protein, results in the internalization of the membrane-associated protein, the binding agent, and the membrane-bound target protein, and wherein the membrane-bound target protein is then degraded. In some embodiments, the membrane-associated protein is recycled to the target cell surface following the internalization of the binding agent. In some embodiments, the membrane-bound target protein is an intracellular protein. In some embodiments, the soluble target protein is an intracellular protein. In some embodiments, the method further comprises modulating an intracellular protein comprising targeting an upstream cell surface receptor with a bispecific or a multispecific antibody. In some embodiments, the intracellular protein is a cell signaling protein. In some embodiments, the cell signaling protein is selected from FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lek, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3 / 6, MKK4 / 7, ASKl,Cot, NIK, Bub, Myt 1, Weel, Casein kinases, PDK1, SGK1, SGK2, SGK3, Aktl, Akt2, Akt3, p90Rsks, p70S6 Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs, Chkl, Chk2, LKB-1, MAPKAPKs, Piml, Pim2, Pim3, IKKs, Cdks, Inks, Erks, IKKs, GSK3a, GSK3P, Cdks, CLKs, PKR, PI3 -Kinase, mTor, SAPK / JNK1,2,3, p38s, PKR, DNA-PK, ATM, or ATR. In some embodiments, the cell signaling receptor is selected from H-Ras, K-Ras, or N-Ras. In some embodiments, the modulating of the intracellular protein comprises downregulating, upregulating, or post- translationally modifying the intracellular protein. In some embodiments, the modulating of the intracellular protein comprises modulating the scaffolding of the intracellular protein.

[0010] Provided herein are methods of degrading a BAFF protein, the method comprising: contacting the BAFF protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the BAFF protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the BAFF protein is internalized with the membrane-associated protein into a target cell and the BAFF protein is degraded.

[0011] Provided herein are methods of degrading an IgG4 protein, the method comprising: contacting the IgG4 protein and a membrane-associated protein with a binding agent; andwherein the binding agent comprises: a first binding domain that specifically binds to the IgG4 protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the IgG4 protein is internalized with the membrane-associated protein into a target cell and the IgG4 protein is degraded.

[0012] Provided herein are methods of degrading a TNFa protein, the method comprising: contacting the TNFa protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the TNFa protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the TNFa protein is internalized with the membrane-associated protein into a target cell and the TNFa protein is degraded.

[0013] Provided herein are methods of degrading a MICA / B protein, the method comprising: contacting the MICA / B protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the MICA / B protein, and a second binding domain that specifically binds to the membrane- associated protein; wherein the MICA / B protein is internalized with the membrane-associated protein into a target cell and the MICA / B protein is degraded.

[0014] Provided herein are methods of degrading an IL-23 protein, the method comprising: contacting the IL-23 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the IL- 23 protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the IL-23 protein is internalized with the membrane-associated protein into a target cell and the IL-23 protein is degraded.

[0015] Provided herein are methods of degrading an IL-1 protein, the method comprising: contacting the IL-1 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the IL-1 protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the IL-1 protein is internalized with the membrane-associated protein into a target cell and the IL-1 protein is degraded.

[0016] Provided herein are methods of degrading an IL-6 protein, the method comprising: contacting the IL-6 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the IL-6 protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the IL-6 protein is internalized with the membrane-associated protein into a target cell and the IL-6 protein is degraded.

[0017] Provided herein are methods of degrading a IFNg protein, the method comprising: contacting the IFNg protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the IFNg protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the IFNg protein is internalized with the membrane-associated protein into a target cell and the IFNg protein is degraded.

[0018] Provided herein are methods of degrading an A0 protein, the method comprising: contacting the A0 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the A0 protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the A0 protein is internalized with the membrane-associated protein into a target cell and the A0 protein is degraded. In some embodiments, the membrane-associated protein is not an Fey receptor. In some embodiments, the A0 protein is an intracellular or extracellular A0 protein.

[0019] Provided herein are methods of degrading a MerTK protein, the method comprising: contacting the MerTK protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the MerTK protein, and a second binding domain that specifically binds to the membrane- associated protein; wherein the MerTK protein is internalized with the membrane-associated protein into a target cell and the MerTK protein is degraded.

[0020] Provided herein are methods of degrading a PD-1 protein, the method comprising: contacting the PD-1 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the PD- 1 protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the PD-1 protein is internalized with the membrane-associated protein into a target cell and the PD-1 protein is degraded.

[0021] Provided herein are methods of degrading a ICOS protein, the method comprising: contacting the ICOS protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the ICOS protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the ICOS protein is internalized with the membrane-associated protein into a target cell and the ICOS protein is degraded.

[0022] Provided herein are methods of degrading a TIM3 protein, the method comprising: contacting the TIM3 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the TIM3 protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the TIM3 protein is internalized with the membrane-associated protein into a target cell and the TIM3 protein is degraded.

[0023] Provided herein are methods of degrading a B7H4 protein, the method comprising: contacting the B7H4 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the B7H4 protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the B7H4 protein is internalized with the membrane-associated protein into a target cell and the B7H4 protein is degraded.

[0024] Provided herein are methods of degrading a RAGE protein, the method comprising: contacting the RAGE protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the RAGE protein, and a second binding domain that specifically binds to the membrane- associated protein; wherein the RAGE protein is internalized with the membrane-associated protein into a target cell and the RAGE protein is degraded.

[0025] Provided herein are methods of degrading an a4 integrin protein, the method comprising: contacting the a4 integrin protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the a4 integrin protein, and a second binding domain that specifically binds to the membrane-associated protein; wherein the a4 integrin protein is internalized with the membrane-associated protein into a target cell and the a4 integrin protein is degraded. In some embodiments, the membrane-associated protein comprises single-pass and multi-pass membrane proteins. In some embodiments, the membrane-associated protein comprises a tissue-type specific protein. In some embodiments, the membrane-associated protein comprises a cell-type specific protein. In some embodiments, the membrane-associated protein comprises an endogenous internalizing receptor or a membrane-associated ubiquitin E3 ligase. In some embodiments, the endogenous internalizing receptor comprises targeting receptors and recycling receptors. In some embodiments, the membrane-associated protein is selected from one or more of the following: HER2, CD30, CXCR7, EPHA2, CCR8, 0X40, CD79B, Nectin- 4, BCMA, integrin 07, IL1RAP, EGFR, CD33, MUC1, ITGB6, CEACAM5, CDH17, CD20, CD22, CD 19, BAFF-R, CD38, TROP2, B7-H3, MARCH- 1, MARCH-8, MARCH-9, GRAIL,Tissue factor, F0LR1, CD45, c-Kit, and TFRC. In some embodiments, the membrane- associated protein comprises IL1RAP. In some embodiments, the membrane-associated protein comprises integrin P7.

[0026] Provided herein are methods of degrading an IL-23 protein, the method comprising: contacting the IL-23 protein and an integrin P7 protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the IL-23 protein, and a second binding domain that specifically binds to the integrin P7 protein; wherein the IL- 23 protein is internalized with the integrin P7 protein into a target cell and the IL-23 protein is degraded.

[0027] Provided herein are methods of degrading an ICOS protein, the method comprising: contacting the ICOS protein and an IL1RAP protein with a binding agent; and wherein the binding agent comprises: a first binding domain that specifically binds to the ICOS protein, and a second binding domain that specifically binds to the IL1RAP protein; wherein the ICOS protein is internalized with the IL1RAP protein into a target cell and the ICOS protein is degraded. In some embodiments, the binding agent comprises a bispecific, or a multi-specific binding agent. In some embodiments, the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, an immunocytokine, an IgG-cytokine, a knob and hole bispecific IgG, a Fc- Fab, a Fc-cytokine, a scFv-Fc-cytokine, a Fab-cytokine, or a knob and hole bispecific Fc-Fab. In some embodiments, the second binding domain comprises a second binding domain variable heavy chain and a second binding domain variable light chain. In some embodiments, the second binding domain comprises a cytokine, or a biologically active fragment thereof, selected from the group comprising CXCL12, CCL1, CCL2, CCL3, CCL3L1, CCL4, CC4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL11, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, vMIPII, and vCXCl. In some embodiments, the second binding domain specifically binds to one membrane-associated protein. In some embodiments, the target cell comprises a microglia cell, endothelial cell, smooth muscle cell, immune cell, plasma cell, B cell, neuronal cell, innate immune cell, lymphocyte, monocyte, granulocyte, regulatory T cell, T cell, effector T cell, activated CD8 T cell, CD8+ T cell, CD4+ T cell, Th 17 cell, macrophage, or dendritic cell. In some embodiments, contacting of the second binding domain to the membrane-associated protein results in the internalization of the membrane-associated protein and the binding agent. In someembodiments, the membrane-associated protein is recycled to the target cell surface following the internalization of the binding agent.

[0028] Provided herein are pharmaceutical compositions, comprising the binding agent(s) disclosed herein, and a pharmaceutically acceptable excipient. Provided herein are methods of treating a disorder in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of treating a disorder in a subject, wherein the method comprises depleting a target protein in the subject by administering to the subject a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. In some embodiments, the target protein is selected from one or more of the following: BAFF, IgG4, TNFa, MICA / B, IL-23, IL-1, IL-2, IL-4, IL-6, IL- 12, IFNg, Ap protein, MerTK, PD-1, ICOS, TIM3, B7H4, RAGE, HLA DQ2.5, GSDMD, TLR7and a4 integrin protein. In some embodiments, the target protein comprises BAFF. In some embodiments, the target protein comprises IgG4. In some embodiments, the target protein comprises TNFa. In some embodiments, the target protein comprises MICA / B. In some embodiments, the target protein comprises IL-23. In some embodiments, the target protein comprises IL-1. In some embodiments, the target protein comprises IL-2. In some embodiments, the target protein comprises IL-4. In some embodiments, the target protein comprises IL-6. In some embodiments, the target protein comprises IL-12. In some embodiments, the target protein comprises IFNg. In some embodiments, the target protein comprises A0 protein. In some embodiments, the target protein comprises MerTK. In some embodiments, the target protein comprises PD-1. In some embodiments, the target protein comprises ICOS. In some embodiments, the target protein comprises TIM3. In some embodiments, the target protein comprises B7H4. In some embodiments, the target protein comprises RAGE. In some embodiments, the target protein comprises HLA DQ2.5. In some embodiments, the target protein comprises GSDMD. In some embodiments, the target protein comprises TLR7. In some embodiments, the target protein comprises a4 integrin. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the disorder comprises a neoplastic disorder, an inflammatory-related disease, an autoimmune disease, a cardiovascular disease, or a neurological disorder. In some embodiments, the neoplastic disorder comprises breast cancer, B cell lymphoma, pancreatic cancer, Hodgkin’s lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin’s B-cell (B-NHL), melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma,glioma, glioblastoma, bladder cancer, or colorectal cancer. In some embodiments, the inflammatory-related disease comprises inflammatory intestinal disease, type 1 diabetes, arthritis, rheumatoid arthritis, psoriasis, lupus, Crohn's disease, colitis, or autoantibody-driven pathologies. In some embodiments, the autoimmune disease comprises SLE, scleroderma, addison disease, celiac disease - sprue, gluten- sensitive enteropathy, dermatomyositis, Graves disease, hashimoto thyroiditis, type 1 diabetes, lupus, alopecia areata, pemphigus vulgaris, inflammatory bowel disease, crohn disease, ulcerative colitis, multiple sclerosis, or myasthenia gravis. In some embodiments, the neurological disorder comprises Parkinson's disease, Alzheimer's disease, a neurodegeneration disease, or multiple sclerosis.

[0029] Provided herein are methods of depleting BAFF in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical(s) disclosed herein. Provided herein are methods of depleting IgG4 in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting TNFa in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting MICA / B in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting IL- 23 in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting IL-1 in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting IL-6 in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting IFNg in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting A0 protein in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting MerTK in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting PD-1 expressing regulatory T cells in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting ICOS expressing T cells in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting TIM3 in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting B7H4 in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting RAGE expressing innate immune cells in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein. Provided herein are methods of depleting a4 integrin in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition(s) disclosed herein.

[0030] In some embodiments, the membrane-bound target protein is an intracellular protein. In some embodiments, the soluble target protein is an intracellular protein. In some embodiments, the method further comprises modulating an intracellular protein comprising targeting an upstream cell surface receptor with a bispecific or a multispecific antibody. In some embodiments, the intracellular protein is a cell signaling protein. In some embodiments, the cell signaling protein is selected from FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lek, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3 / 6, MKK4 / 7, ASK 1, Cot, NIK, Bub, Myt 1, Weel, Casein kinases, PDK1, SGK1, SGK2, SGK3, Aktl, Akt2, Akt3, p90Rsks, p70S6 Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs, Chkl, Chk2, LKB-1, MAPKAPKs, Piml, Pim2, Pim3, IKKs, Cdks, Inks, Erks, IKKs, GSK3a, GSK3P, Cdks, CLKs, PKR, PI3-Kinase, mTor, SAPK / JNK1,2,3, p38s, PKR, DNA-PK, ATM, or ATR. In some embodiments, the cell signaling receptor is selected from H-Ras, K-Ras, or N-Ras. In some embodiments, the modulating of the intracellular protein comprises downregulating, upregulating, or post-translationally modifying the intracellular protein. In some embodiments, the modulating of the intracellular protein comprises modulating the scaffolding of the intracellular protein.INCORPORATION BY REFERENCE

[0031] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The novel features of the inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present inventive concepts will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concepts are utilized, and the accompanying drawings of which:

[0033] FIG. 1 depicts the selection of receptors based on cell-specific enrichment to achieve cell-type selective targeting achieving the most impact based on cell and tissue selectivity and disease space.

[0034] FIG. 2 depicts the use of a bispecific binding agent (e.g., antibody) targeting an ICOS protein and an endogenous internalizing receptor that is selectively enriched on a cytotoxic T-cell (e.g., interleukin-1 receptor accessory protein or IL1RAP) to induce the selective internalization and lysosomal degradation of the target protein, ICOS, in cytotoxic T- cells relative to T-regulatory cells.

[0035] FIG. 3A depicts the use of a bispecific binding agent (e.g., antibody) to induce the selective internalization and lysosomal degradation of the target protein, PD-1. The diagram depicts an exemplary bispecific binding agent targeting a PD-1 protein and a cell-type specific endogenous internalizing receptor (e.g., 0X40, CD30, or CCR8) that is selectively enriched on the surface of T cells (e.g., T-regulatory cells).

[0036] FIG. 3B shows PD-1 protein cell-surface levels on T cells (e.g., human CD4+ T cells) after treatment with an exemplary bispecific binding agent which targets the cell-surface protein, PD-1, and an internalizing receptor e.g., CD30) that is selectively enriched on the surface of the T cells. Results indicate the level of surface PD-1 as determined by fluorescence intensity; treatments include an isotype control, Epi692, two monospecific binding agentswhich bind only PD-1 or CD30, Epi3086 and Epi2862, respectively, and the exemplary bispecific binding agent which binds both PD-1 and CD30, Epi 2853.FIG. 4 illustrates the use of a bispecific antibody to preferentially bind a particular cell lineage within a population as opposed to a non-specific neutralizing antibody. Monovalent antibodies would not have the ability to localize preferentially to a particular cell lineage due the inability to bind to a cell surface marker on the target cell and the protein target.

[0037] FIG. 5A depicts the flow cytometry gating strategy to isolate regulatory T cells (Tregs) from T effector cells (Teffs). Tregs are high in FoxP3 and CD25. Gating a heterogeneous T cell population by these results in Treg cells emerging as a separate population from Teff cells.

[0038] FIG. 5B depicts flow cytometry data demonstrating cell-surface enrichment of CD30 preferentially on Tregs vs Teffs. While PD-1 is present on both cell types, CD30 is enriched preferentially in the Treg population.

[0039] FIG. 6A shows flow cytometry results taken from populations of CD4+ T cells stained for cell surface PD-1 after treatment with varying concentrations of bispecific antibodies to CD30 and / or PD-1. Cells receiving bispecifics to both PD-1 and CD30, a degrader, demonstrated reduced levels of surface PD-1 in a dose-dependent manner.

[0040] FIG. 6B illustrates the results from the 200ng / mL treatment groups in FIG. 6A. The groups treated with the dual PD-1 / CD30 binders at this concentration were lowly PD- 1+, while single armed binders to the degrader itself had no effect on PD-1 positivity.

[0041] FIG. 7A displays flow cytometry data demonstrating surface degradation of PD-1 in a CD30 / PD-1 bispecific antibody-dose dependent manner. This was performed along with intracellular staining to confirm internalization and degradation of PD-1 on CD4+ T cells.

[0042] FIG. 7B displays flow cytometry data, following a permeabilization step, clearly demonstrating intracellular degradation of PD-1 in a CD30 / PD-1 bispecific antibody-dose dependent manner. This was performed along with surface staining to confirm internalization and degradation of CD4+ T cells.

[0043] FIG. 8A displays flow cytometry data demonstrating surface degradation of PD-1 in a OX40 / PD-1 bispecific antibody-dose dependent manner. This was performed along with intracellular staining to confirm internalization and degradation of PD-1 on CD4+ T cells.

[0044] FIG. 8B displays flow cytometry data, following a permeabilization step, clearly demonstrating intracellular degradation of PD-1 in a OX40 / PD-1 bispecific antibody-dose dependent manner. This was performed along with surface staining to confirm internalization and degradation of CD4+ T cells.

[0045] FIG. 9 are graphical results of a bioinformatics screen to identify cell-surface markers preferentially enriched on Tregs vs Teffs. Bioinformatics identifies CCR4, CCR8 and TNFR2 as some Treg enriched genes.

[0046] FIG. 10A is the graphical result from flow cytometry experiments similar to FIGS. 7-8 demonstrating Treg preferential reduction of PD-1 levels using EpiTACs targeting PD-1 and CCR4.

[0047] FIG. 10B is the graphical result from flow cytometry experiments similar to FIGS. 7-8 demonstrating Treg preferential reduction of PD-1 levels using EpiTACs targeting PD-1 and CCR8.

[0048] FIG. 11A displays the flow cytometry gating scheme to select for Tregs among a heterogenous population. One group of cells in the upper right quadrant is enriched for both FoxP3 and CD25.

[0049] FIG. 11B displays additional flow cytometry data from FIG. 11 A. This graph shows that 56.2% of the T effector cells in their population were CD25+

[0050] FIG. 11C displays a separate flow cytometry result from the T cells used in FIGS. 11 A-B. This graph shows pSTAT5 signaling resulting from treatment with either WT IL-2 or IL-2 mutein was only elicited in CD25+ cells.

[0051] FIG. 12A displays a similar flow cytometry result as in FIG. 1 IB, although this time on display is a population of T effector cells. The results show that 99% of the Teffs are CD25-.

[0052] FIG. 12B This graph is similar to that found in FIG.11C. It shows no pSTAT5 signaling resulting from treatment with either WT IL-2 or IL-2 mutein in CD25- cells (Teff).

[0053] FIG. 13A is a flow cytometry result demonstrating the activation of pSTAT5 by EpiTACs composed of either WT IL-2 or IL-2 mutein along with an arm targeting PD-1. The results show pSTAT5 can be activated by the bispecific.

[0054] FIG. 13B is a flow cytometry result demonstrating the reduction of PD-1 surface signaling by EpiTACs composed of either WT IL-2 or IL-2 mutein along with an arm targeting PD-1. The results show targeting PD-1 alone or with a PD-l / IL-2 (WT / Mut) can cause degradation of PD-1.

[0055] FIG. 14 depicts the use of a bispecific binding agent (e.g., antibody) targeting a RAGE protein and an endogenous internalizing receptor specific to inflamed tissues to induce the selective degradation of RAGE in a tissue-specific manner.

[0056] FIG. 15A is an illustration of function of a bispecific binding agent designed to target IL-23 and an additional target like an endogenous internalizing receptor on cancer cells, specific T cells, or specific innate immune cells which are located within a specific tissue-type.

[0057] FIG. 15B shows results from an extracellular detection kit where simultaneous binding of CD71 and an extracellular soluble protein through bispecific or multispecific antibodies (e.g., exemplary binding agents of the present disclosure) mediated the internalization of the extracellular soluble protein (IgG4 or IL23).

[0058] FIG. 15C shows results from an extracellular detection kit where cells were treated with bispecifics for alfa (against alfa tagged IgG4) and either EphA2 or CD71 degrading receptors. The results show that this combination of binding components results in the degradation of extracellular IgG4 in a dose-dependent manner.

[0059] FIG. 16A shows results from an extracellular and intracellular detection kit. Binders to CD71 and alfa result in reduced extracellular alfa-tagged IgG, but no increase of intracellular proteins. Thus, degradation and not merely internalization is established.

[0060] FIG. 16B is a representative illustration of an EpiTAC bispecific antibody. It is depicted in this example as having three binding locations for two targets.

[0061] FIG. 17A is a graph showing the results of an in vitro degradation experiment utilizing a dual CD71 and either alfa or direct IgG4 (Mal8) binding sites. Binding to the alfa tags or direct binding to IgG4s were both effective at degrading extracellular IgG4.

[0062] FIG. 17B is a graph showing the results of an in vivo degradation experiment utilizing a dual CD71 and either alfa or direct IgG4 (Mal8) binding sites. Similarly, binding to the alfa tags or direct binding to IgG4s were both effective at degrading extracellular IgG4.

[0063] FIG. 18A is a graph showing the results of an in vitro internalization experiment utilizing a fluorescently labeled IgG4 and a suite of EpiTACs. Only the treatment conditions with dual IgG4 and CD71 resulted in increased internalization.

[0064] FIG. 18B is a graph showing the results of an in vitro extracellular removal experiment utilizing a HiBiT labeled IgG4 and a suite of EpiTAC configurations. Only the treatment conditions with dual IgG4 and CD71 resulted in decreased levels of IgG4 in the supernatant.

[0065] FIG. 19 is an illustration of the different EpiTAC configurations utilized in these studies. Some variants include single-armed Alfa binders, and multiple variants utilizing CD71 as the degrader and either Alfa or Mai to target the IgG4. Pali is a negative control.

[0066] FIG. 20A is a graph showing the serum concentrations of IgG4 remaining after treatment of mice with various bispecific configurations, using a HiBiT assay. Bispecifics targeting Alfa IgG4 and CD71 resulted in near clearance of IgG4.

[0067] FIG. 20B is a graph showing the serum concentrations of IgG4 remaining after treatment of mice with various bispecific configurations. Bispecifics targeting IgG4 directly and CD71 resulted in near clearance of IgG4.

[0068] FIG. 21 is an illustration of the use of a target agnostic bispecific that binds to any alfa-tagged receptor, as well as soluble IgG4. Use of this universal degrader can allow one molecule to be compatible with a multitude of cell surface degrading receptors.

[0069] FIG. 22 is a graph displaying the results of a universal “alfa screen” to detect relative levels of known degrading receptors on the surface of HeLA cells. About sixty different cell-surface candidates were identified with this screen.

[0070] FIG. 23 is a graph displaying the results of a screen of those sixty candidate binders revealed in FIG. 22 with the goal of narrowing the choices of best candidates. Fifteen prime candidates were identified for their efficacy in decreasing extracellular IgG4.

[0071] FIG. 24 is a graph displaying the results of another screen of those sixty revealed in FIG. 22 to select the best performing binder candidate. The results of this experiment identified seven promising targets.

[0072] FIG. 25 is a graph displaying the results of another screen of those sixty revealed in FIGS. 22 to select the best performing binder candidate. The results of this experiment identified nine promising targets.

[0073] FIG. 26 is a chart displaying summarized results of this universal binder screen. In sum, fifteen degraders emerged as the best candidates, each with different cellular functions and expression patterns.

[0074] FIG. 27A depicts the use of an EpiTAC bispecific agent targeting Ap aggregated protein (e.g., amyloid beta fibrils) and an endogenous internalizing receptor to degrade extracellular Ap in a non-inflammatory manner.

[0075] FIG. 27B shows another depiction of the use of a bispecific binding agent (e.g., antibody) targeting Ap aggregated protein (e.g., amyloid beta fibrils) and an endogenous internalizing receptor (e.g., non-Fc internalizing receptors located on microglia or endothelial cells) to induce the internalization and lysosomal degradation of the aggregated target protein, often in an inflammatory manner.

[0076] FIG. 27C shows the uptake of amyloid beta fibrils by human Non-Small Cell Lung Cancer cells e.g., H1975 cells) after treatment with an exemplary bispecific binding agentwhich targets the amyloid beta fibrils and an internalizing receptor (e.g., CD71). Results were graphed as fluorescence intensity in H1975 cells; amyloid beta fibrils were labeled with a pH sensitive dye (pHrodo); treatments include a monospecific binding agent which binds only amyloid beta fibrils (e.g., aducanumab or bapineuzumab), and the exemplary bispecific binding agent which binds both the amyloid beta fibrils (e.g., aducanumab or bapineuzumab) and CD71.

[0077] FIG. 28A shows a graph displaying the results of an Ap internalization assay. Human monocytes were cultured with pHrodo labeled Ap fibrils in the presence of the indicated bispecific antibodies. Internalization was monitored by fluorescent signal accumulated within cells (integrated intensity). Bispecific antibodies targeting Ap and either CD71 or CD 163 mediated the amount of Ap internalized over the period of the assay.

[0078] FIG. 28B is a graph displaying results from a parallel experiment to Fig. 28A. Cells from that experiment were harvested at 19 and 72 hours and the concentration of internalized Ap lysates were quantified via ELISA assays. Bispecific antibodies targeting Ap and CD163 resulted in an increase of intracellular Ap at 19 hours (internalization), and a subsequent decrease of Ap at 72 hours, suggesting degradation of the internalized protein.

[0079] FIG. 29A shows a graph displaying Luminex cytokine profiling results. The concentration of TNF-a in the supernatant after bi specific antibody treatment of the monocytes in FIG. 28 was significantly lower in groups treated with Ap / CD71 bispecific antibodies vs the monovalent Ap positive control antibody.

[0080] FIG. 29B shows a graph displaying Luminex cytokine profiling results. The concentration of IL-6 in the supernatant after bispecific antibody treatment of the monocytes in FIG. 28 was significantly lower in groups treated with Ap / CD71 bispecific antibodies vs the monovalent Ap positive control antibody.

[0081] FIG. 29C shows a graph displaying Luminex cytokine profiling results. The concentration of IL- 10 in the supernatant after bispecific antibody treatment of the monocytes in FIG. 28 was significantly lower in groups treated with Ap / CD71 bispecific antibodies vs the monovalent Ap positive control antibody.

[0082] FIG. 29D shows a graph displaying Luminex cytokine profiling results. The concentration of IL-ip in the supernatant after bispecific antibody treatment of the monocytes in FIG. 28 was significantly lower in groups treated with Ap / CD71 bispecific antibodies vs the monovalent Ap positive control antibody.

[0083] FIG. 29E shows a graph displaying Luminex cytokine profiling results. The concentration of IL-33 in the supernatant after bispecific antibody treatment of the monocytesin FIG. 28 was significantly lower in groups treated with Ap / CD71 bispecific antibodies vs the monovalent Ap positive control antibody.

[0084] FIG. 29F shows a graph displaying Luminex cytokine profiling results. The concentration of VEGF in the supernatant after bi specific antibody treatment of the monocytes in FIG. 28 was significantly lower in groups treated with Ap / CD71 bispecific antibodies vs the monovalent Ap positive control antibody.

[0085] FIG. 30A shows a graph displaying Luminex cytokine profiling results. The concentration of TNF-a in the supernatant after bispecific antibody treatment of monocytes was significantly lower in groups treated with Ap / CD163 bispecific antibodies vs the monovalent Ap positive control antibody.

[0086] FIG. 30B shows a graph displaying Luminex cytokine profiling results. The concentration of IL-6 in the supernatant after bispecific antibody treatment of monocytes was significantly lower in groups treated with Ap / CD163 bispecific antibodies vs the monovalent Ap positive control antibody.

[0087] FIG. 30C shows a graph displaying Luminex cytokine profiling results. The concentration of IL-ip in the supernatant after bi specific antibody treatment of monocytes was significantly lower in groups treated with Ap / CD163 bispecific antibodies vs the monovalent Ap positive control antibody.

[0088] FIG. 31 shows a graph displaying ELISA assay results. The concentration of Ap present in the supernatant of monocyte cultures treated with an bispecific antibodies targeting Ap and CD 163. Treatment with this bispecific resulted in a decrease of extracellular Ap over the period of the assay.

[0089] FIG. 32A depicts the use of a bispecific binding agent (e.g., antibody) targeting an endogenous internalizing receptor (e.g., degrader receptor) and in intracellular protein (e.g., an endosomal-associated protein, such as TLR7). This is achieved through the internalization of a bispecific antibody, hence, allowing the accessibility of the intracellular target by said bispecific antibody, inducing the lysosomal degradation of the intracellular protein.

[0090] FIG. 32B shows another depiction of the use of a bispecific binding agent (e.g., antibody) targeting an endogenous internalizing receptor (e.g., degrader receptor) and in intracellular protein (e.g., an endosomal-associated protein, such as TLR7). This is achieved through the internalization of a bispecific antibody, hence, allowing the accessibility of the intracellular target by said bispecific antibody, inducing the lysosomal degradation of the intracellular protein.

[0091] FIG. 32C shows the levels of TLR7 signaling in Hek-blue hTLR7 reporter cells after treatment with an exemplary bispecific binding agent which targets an intracellular protein, TLR7, and an internalizing receptor (e.g., degrader receptor). Results were graphed as absorbance; treatments include an isotype control, pali IgG, a monospecific binding agent which binds only TLR7, and the exemplary bispecific binding agent which binds both the intracellular target protein e.g., TLR7) and an internalizing receptor (e.g., degrader receptor).

[0092] FIG. 33 is an illustration summarizing events occurring due to TLR7 agonism. An agonist, like the small molecule R-848, is internalized and binds with TLR7. This triggers NFkB and other transcription machinery to turn on many genes, including those involved with inflammation.

[0093] FIG. 34 is an illustration of what is predicted to occur if TLR7 signaling is inhibited. For instance, proteins that are downstream gene products of TLR7, such as inflammatory cytokines, will be expected to decrease in expression due to successful TLR7 degradation.

[0094] FIG. 35A is graph depicting a luminescence experiment using a TLR7 reporter cell line, the small molecule R-848 at 20ng / mL and bispecific antibodies at the indicated concentrations. Only the treatment groups receiving bispecifics that bind CD71 and TLR7 exhibited reduced TLR7 activity in response to 20ng / mL R-848 co-treatment.

[0095] FIG. 35B is graph depicting a luminescence experiment using a TLR7 reporter cell line, the small molecule R-848 at 20ng / mL and bispecific antibodies at the indicated concentrations. At lower concentrations, only the treatment groups receiving bispecifics that bind CD71 and TLR7 displayed reduced TLR7 activity in response to 20ng / mLR-848 cotreatment.

[0096] FIG. 36A is a graph displaying the results of a Luminex cytokine experiment. B- cells stimulated with R-848 and co-treated with CD19 / TLR7 bispecifics demonstrated reduced levels of proinflammatory cytokines, like IL-6 as shown.

[0097] FIG. 36B is a graph displaying the results of a Luminex cytokine experiment. B- cells stimulated with R-848 and co-treated with CD19 / TLR7 bispecifics demonstrated reduced levels of proinflammatory cytokines, like TNF-a as shown.

[0098] FIG. 36C is a graph displaying the results of a Luminex cytokine experiment. B- cells stimulated with R-848 and co-treated with CD19 / TLR7 bispecifics demonstrated reduced levels of proinflammatory cytokines, like MIPlb as shown.

[0099] FIG. 37 shows tumor cell viability (e.g., % survival) for patient derived organoids (PDOs) from colorectal cancers (CRCs) after treatment with an exemplary bispecific binding agent which targets a membrane-associate protein, EGFR, and an internalizing receptor (e.g.,CD71) and membrane expressed E3 ligase (e.g, RNF43). The CRC PDOs express elevated EGFR and other oncogenic mutations (e.g., KRAS mutations). The exemplary bispecific binding agent shows suppression of CRC PDO tumor growth independent of oncogenic mutations (e.g., KRAS mutations). Tumor cell viability was measured 5 days post-treatment with a CellTiter-Glo® assay. Data points represent the average of three replicates, and % survival was calculated relative to vehicle (out of 100%) using background removal.

[0100] FIG. 38 shows a western blot of total EGFR, ERK and p-ERK in KRAS mutant CRC cell line after treatment with an exemplary bispecific binding agent which targets a membrane-associate protein, EGFR, and an internalizing receptor (e.g., CD71) or and E3 ligases (eg RNF43).

[0101] FIG. 39A shows a western blot of EGFR / pEGFR for non-small cell lung cancer (NSCLC) tumors harvested after 72 hrs of treatment (lOmpk dose) with an exemplary bispecific binding agent which targets a membrane-associate protein, EGFR, and an internalizing receptor (e.g., CD71).

[0102] FIG. 39B shows mutant EGFR tumor protein levels for non-small cell lung cancer (NSCLC) cells after 72 hrs of treatment (lOmpk dose) with an exemplary bispecific binding agent which targets a membrane-associate protein, EGFR, and an internalizing receptor (e.g., CD71). The top panel depicts the quantification of normalized EGFR / pEGFR WB levels. The bottom panel depicts the quantification of normalized ERK / pERK AlphaLISA.

[0103] FIG. 40 is an illustration of the activation mechanism governing c-Kit and its interactions with stem cell factor (SCF). Gain of function mutations in the receptor can lead to self-activation and aberrant downstream signaling.

[0104] FIG. 41 is a chart of the results from informatics data. The results show twelve possible degraders that are enriched in GIST cells, detectable by FACs and / or mass spec.

[0105] FIG. 42 is a graphical workflow of the efforts to identify a bispecific antibody configuration with c-Kit cell surface removal and tumor growth inhibition. It begins with choosing c-Kit binders with valuable properties. Next, the identification of degraders that synergize with c-Kit, Last, assays to demonstrate superior activity in a clinically relevant model.

[0106] FIG. 43 is a chart displaying six different binding motifs of c-Kit that can be employed on the GIST bispecific. They all have a KD of less than 50nm, induce cell surface removal. Most binders, except 2D1 and 3G1 cause in vitro growth inhibition.

[0107] FIG. 44 is a graph illustrating the results of a cell growth assay with treatments that include bispecifics with the CDX0168 c-Kit degrader identified in FIG. 43, and variousdegrading receptors. MRC2 binders were identified as the most potent binding partner to CDX0168 in terms of reduction of cell proliferation on par with the imatinib control.

[0108] FIG. 45 displays a series of western blots showing a change in the expression levels of Kit in cells treated with varying concentrations of bispecific pairs containing either the CDX Al c-Kit binder or 3G1 binding arm. Global concentrations of Kit were reduced markedly when treated with bispecifics containing the G7V binder, paired with either CDX Al or 3G1.

[0109] FIG. 46A is a graph of in vitro cell growth data in response to treatment with bispecifics containing both CDX Al and G7V arms. Cells treated with this bispecific configuration demonstrated lagged growth over a span of 10 days, potentially better to the imatinib positive control.

[0110] FIG. 46B is a graph of in vitro cell growth data in response to treatment with bispecifics containing only one of the CDX Al and G7V arms. Cells treated with this binder configuration demonstrated no growth differences over a span of 10 days, while the imatinib positive control showed efficacy.

[0111] FIG. 47A is a graph that illustrates the results of a cell surface c-Kit detection assay. GIST cells were exposed to various configurations of bispecifics including single armed c-Kit binders to dual armed c-Kit / MRC2 binders. Results are reported as %Kit cell surface removal. The CDXA1 / G7V combination uniquely appears to degrade more c-Kit than the single armed binder is capable of.

[0112] FIG. 47B is a chart displaying the different c-Kit binder clones used in Fig.47A, as well as the internalizing receptor that results in target degradation.

[0113] FIG. 48 is a series of western blot results depicting the fate of the degrading receptor once engaged by the MRC2 / c-Kit bispecific. Although there is evidence of c-Kit degradation within 24 hours, the levels of MRC2 did not change when using the G7V binding arm. The degrader with the best efficacy, CDX A1 / G7V, appeared to also be the configuration that does not result in MRC2 degradation.

[0114] FIGs. 49A-C are a series of graphs illustrating results from a flow cytometry experiment comparing CCR5 positivity in T cells treated with bispecifics of varying configurations, including the CCR5 / CD71 pair. Across all T cells, including CD4s and CD8s, treatment with this bispecific pairing resulted in significant CCR5 loss in the T cell population.

[0115] FIG. 50 is a diagram summarizing the spectrum of differences between T cell lineages with Teff and Tregphenotypes appearing each end of the spectrum. There exists anintermediate lineage, TEM, that are more likely to be present at a target tissue and may be a more physiologically relevant target than Tregs.

[0116] FIG. 51 A is a graph illustrating results from a flow cytometry experiment comparing CCR5 positivity in TEM treated with bi specifics of varying configurations, including the CCR5 / CD71 pair. CD8+TEM cells treated with this bispecific pairing resulted in significant CCR5 loss in the T cell population.

[0117] FIG. 51B is a graph illustrating results from a flow cytometry experiment comparing CCR5 positivity in TEM treated with bi specifics of varying configurations, including the CCR5 / CD71 pair. Total TEM cells treated with this bispecific pairing resulted in significant CCR5 loss in the T cell population.

[0118] FIG. 52A is a western blot illustrating the results of an alfa-tagged protein degradation experiment. Cells were transfected with Alfa-tagged CCR5, then treated with bispecific antibodies of various configurations. Epi4025, the binder that targets CD71 and Alfa, demonstrated the most degradation of all of the binders tested.

[0119] FIG. 52B is data from FIG. 52A represented in bar graph form with band sizes quantified. Alfa-tagged proteins were degraded at varying degrees depending on treatment type. Epi4025, the binder that targets CD71 and Alfa, demonstrated the most degradation of all the binders tested.

[0120] FIG. 53 is an illustration of the universal degrader system in use. Alfa-tagged receptors displayed on a cell of interest can be targeted by a multitude of binding pairs containing an Alfa-binding component. High throughput screens can be conducted using the same binder for all conditions.

[0121] FIG. 54 is a graph depicting the results of a alfa-tag screen for degrading receptors appropriate for targeting and degrading EGFR.DETAILED DESCRIPTION

[0122] To date, most targeted protein degraders are heterobifunctional small molecule degraders that recruit intracellular E3 ubiquitin ligases to a target of interest, which induces ubiquitination of the target protein and its subsequent degradation by the proteasome. However, due to their intracellular mechanism of action, these degraders are largely limited to targeting intracellular proteins for degradation. Furthermore, targeted protein degraders have had limited targeting capability to specific tissues and cell types. Hence, there continues to exist a need fortargeted protein degraders that efficiently and selectively induce the degradation of a target protein.

[0123] In recent years, various immune proteins and immune cells have been identified. Disruptions in these proteins and / or cells (e.g., mutations) have been implicated in autoimmune disease development. However, therapies targeting proteins involved in the immune response often result in general immune suppression due to lack of specificity for tissues and cell types, which can lead to infections and other serious complications. Therefore, targeting regulatory immune proteins and / or cells through targeted protein degradation (either to specific sites of inflammation, specific tissue types, and / or specific cell types) could be a promising therapeutic strategy for treating different autoimmune diseases, as well as limiting general immune suppression mediated by neutralizing proteins. Thus, there is a need for targeted protein degradation which can degrade not only extracellular proteins (e.g., membrane-bound, soluble, protein aggregates, etc.) but also tissue specific and cell-type specific proteins. Selecting receptors enriched on different types of T-cells for targeted inhibition is a crucial approach in managing autoimmune diseases and infections, where the balance of immune response is pivotal. In autoimmune diseases, an overactive immune response against the body's own tissues causes damage, whereas in infections, a robust immune response is required to eliminate pathogens. A key to this selective inhibition lies in the selective targeting of cells based on understanding the distinct roles and receptor profiles of different T-cell subsets, such as CD8 T-cells and T-regulatory (Treg) cells.

[0124] CD8 T-cells, also known as cytotoxic T-cells, are primarily involved in the direct destruction of infected or malignant cells. They play a critical role in controlling infections by recognizing and killing cells infected with viruses or other pathogens. However, in autoimmune diseases, these cells can mistakenly target and destroy healthy tissues. In contrast, Treg cells are responsible for maintaining immune tolerance and preventing autoimmune responses. They suppress the overactivation of the immune system and prevent it from attacking the body's own cells. By identifying and targeting receptors that are uniquely enriched or activated in these two cell populations, it is possible to modulate their activities selectively. For instance, inhibiting a receptor that is predominantly expressed on CD8 T-cells could dampen their activity in autoimmune conditions without substantially affecting the beneficial suppressive functions of Treg cells. Conversely, enhancing Treg functions or survival through specific receptor targeting could bolster their role in controlling autoimmunity while preserving the necessary immune responses against infections. This receptor-based selective modulationprovides a pathway to fine-tune the immune system's response, aiming to reduce harmful inflammation in autoimmune diseases while maintaining the body's ability to fight infections.

[0125] Targeted protein degradation by the binding agent(s) of the present disclosure aims to selectively remove intracellular proteins, soluble proteins, or membrane-bound (e.g., membrane-associated) proteins from specific cell-types. Soluble proteins can refer to proteins which are found outside cells in extracellular spaces (e.g., extracellular proteins). Intracellular proteins can be challenging to target because they are not directly accessible from outside the cell. However, cells possess natural protein internalization and degradation machinery, such as the endosomal-lysosomal system or ubiquitin-proteasome system (UPS), which regulate protein turnover. The binding agent(s) of the present disclosure harness these pathways for targeted protein degradation by selectively engaging these degradation systems and consequently triggering the degradation of targeted proteins (e.g., intracellular, soluble, and / or membrane-bound proteins).

[0126] The disclosure provided herein demonstrates the development of a targeted protein degradation platform, which includes binding agents that bind both membrane-associated proteins enriched on the surface of specific cell types, as well as therapeutically relevant cell surface proteins, extracellular soluble proteins, and / or intracellular proteins, for receptor- mediated internalization and subsequent lysosomal degradation of the therapeutically relevant target proteins. In some non-limiting exemplary embodiments, the therapeutically relevant target protein is an ICOS protein (FIG. 2), a PD-1 protein (FIG. 3A-B), a RAGE protein (FIG. 14), an IL-23 protein (FIG. 15A-B), an Ap aggregated protein (FIG. 27A-C), a TLR7 protein (FIG. 32A-C), or an EGFR protein (FIG. 37).I.METHODS OF DEGRADING

[0127] Provided herein is a method of degrading a target protein, the method comprising contacting the target protein and a membrane-associated protein with a binding agent, wherein the binding agent comprises: a first binding domain that specifically binds to the target protein, and a second binding domain that specifically binds to the membrane-associated protein, wherein the membrane-associated protein is associated with a target cell; and wherein the contacting of the target protein and the membrane-associated protein with the binding agent leads to degradation of the target protein. In some embodiments, the binding agent comprises a bispecific antibody (e.g., EpiTACs). In some embodiments, the first binding domain can refer to a target binding arm of the bispecific antibody. In some embodiments, the second binding domain can refer to a degrader binding arm of the bispecific antibody. In someembodiments, the first binding domain (e.g., the target binding arm) together with the second binding domain (e.g., the degrader binding arm) localize degradation of extracellular and membrane targets to disease tissue, sparing normal tissue and increasing therapeutic efficacy.1. Binding agent

[0128] Provided herein is a method of degrading a target protein, the method comprising contacting the target protein and a membrane-associated protein with a binding agent. In some embodiments, the binding agent comprises: a first binding domain that specifically binds to the target protein, and a second binding domain that specifically binds to the membrane-associated protein.

[0129] Binding agents of the disclosure include, without limitation, binding agents wherein the first binding domain and the second binding domain are each independently selected from an antibody (or half of an antibody), a nanobody, or a minibody, a Fab fragment, a single chain variable fragment (scFv), and a single domain antibody (sdAb), or a functional fragment thereof. These two binding domains can be the same type of molecule, or different. For example, binding agents of the disclosure include, without limitation, multispecific binding agents having an IgG that binds a membrane-associated internalizing or degrading protein, and an scFv domain that binds a cell surface receptor. The binding domains of the multispecific binding agent can be connected through covalent bonds, non-covalent interactions, or a combination thereof.

[0130] The binding agent can generally take the form of a protein, glycoprotein, lipoprotein, phosphoprotein, and the like. Some binding agents of the disclosure take the form of multispecific antibodies, bispecific antibodies or antibody derivatives. In some embodiments, the binding agent comprises an antibody. In some embodiments, the binding agent comprises a multispecific antibody. In some embodiments, the binding agent comprises a bispecific antibody. In some embodiments, the binding agent comprises an IgG antibody. In some embodiments, the binding agent comprises a multispecific IgG antibody. In some embodiments, the binding agent comprises a knob and hole bispecific IgG. In some embodiments, the binding agent is not an antibody-drug conjugate (“ADC”). In some embodiments, the binding agent comprises a bispecific binding agent. In some embodiments, the binding agent comprises a bispecific antibody. In some embodiments, the binding agent comprises a bispecific diabody. In some embodiments, the binding agent comprises a bispecific Fab2. In some embodiments, the binding agent comprises a bispecific camelid antibody. In some embodiments, the binding agent comprises a bispecific peptibody scFv-Fc. In someembodiments, the binding agent comprises Fc-Fab. In some embodiments, the binding agent comprises a knob and hole bispecific Fc-Fab. In some embodiments, the target protein binding domain is selected from the group consisting of a half antibody, a nanobody, or a minibody, a F(ab’)2 fragment, a Fab fragment, a single chain variable fragment (scFv), and a single domain antibody (sdAb), or a functional fragment thereof. The binding domains may together take the form of a bispecific antibody, a bispecific diabody, a bispecific camelid antibody or a bispecific peptibody, and the like. Antibody derivatives need not be derived from a specific wild type antibody. For example, one can employ known techniques such as phage display to generate and select for small proteins having a binding domain similar to an antibody complementaritydetermining region (CDR). In some embodiments, the antigen-binding moiety includes an scFv. The binding domain can also be derived from a natural or synthetic ligand or receptor, whether soluble or membrane-bound, that specifically binds to the receptor protein. The binding domain can also be derived from a natural or synthetic ligand or receptor, whether soluble or membrane-bound, that specifically binds to a target protein.

[0131] Multispecific antibodies can be prepared by known methods. Embodiments of the disclosure include “knob-into-hole” bispecific antibodies, wherein the otherwise symmetric dimerization region of a bispecific binding agent is altered so that it is asymmetric. For example, a knob-into-hole bispecific IgG that is specific for antigens A and B can be altered so that the Fc portion of the A-binding chain has one or more protrusions (“knobs”), and the Fc portion of the B-binding chain has one or more hollows (“holes”), where the knobs and holes are arranged to interact. This reduces the homodimerization (A-A and B-B antibodies) and promotes the heterodimerization desired for a bispecific binding agent. See, e.g., Y. Xu et al., mAbs (2015) 7(1):231-42. In some embodiments, the bispecific binding agent has a knob-into- hole design. In some embodiments, the “knob” comprises a T336W alteration of the CH3 domain, i.e., the threonine at position 336 is replaced by a tryptophan. In some embodiments, the “hole” comprises one or a combination of T366S, L368A, and Y407V. In some embodiments, the “hole” comprises T366S, L368A, and Y407V.

[0132] In some embodiments, the multispecific binding agent comprises an FcRn receptor recognition domain, to promote return of the bi specific binding agent to the extracellular space if the bispecific binding agent is internalized.

[0133] The present disclosure provides a binding agent, comprising a first binding domain and a second binding domain. The first binding domain can comprise an “arm” of an antibody (e.g., a binding agent disclosed herein). In some embodiments, the first binding domain comprises an anti-receptor protein arm of the binding agent disclosed herein. The secondbinding domain can comprise an “arm” of an antibody (e.g., a binding agent disclosed herein). In some embodiments, the second binding domain comprises an anti-target protein arm of the binding agent disclosed herein. In the context of bispecific antibodies (e.g., binding agents disclosed herein), an arm of an antibody, such as an arm of the binding agents disclosed herein, can refer to one of the two binding specificities incorporated into the antibody molecule / binding agent. For example, bispecific antibodies are engineered to simultaneously target two different antigens, unlike traditional antibodies that typically recognize a single antigen, and each antibody arm of a bispecific antibody is designed to bind to a specific antigen. For instance, if we consider a bispecific antibody with one arm targeting antigen A (e.g., a receptor protein) and the other arm targeting antigen B (e.g., a target protein), each arm will have its own antigen-binding site. This allows the bispecific antibody (e.g., bispecific antireceptor protein and anti-target protein binding agents disclosed herein) to simultaneously bind to both antigens, likely bringing them in close proximity to each other.

[0134] In some embodiments, the first binding domain comprises a first light chain constant region. In some embodiments, the second binding domain comprises a second light chain constant region. In some embodiments, the first light chain constant region or the second light chain constant region, or a combination thereof comprises a kappa light chain constant region or functional fragment thereof, a lambda light chain constant region or functional fragment thereof, or a combination thereof. In some embodiments, the first binding domain comprises a Fab or a scFv. In some embodiments, the second binding domain comprises a Fab or a scFv.

[0135] In some embodiments, the first binding domain comprises one or more heavy chain constant regions. In some embodiments, the second binding domain comprises one or more heavy chain constant regions. In some embodiments, the one or more heavy chain constant regions selected from the group consisting of IgGl heavy chain constant region or functional fragment thereof, IgG2 heavy chain constant region or functional fragment thereof, IgG3 heavy chain constant region or functional fragment thereof, IgGAl heavy chain constant region or functional fragment thereof, IgGA2 heavy chain constant region or functional fragment thereof, IgG4 heavy chain constant region or functional fragment thereof, IgJ heavy chain constant region or functional fragment thereof, IgM heavy chain constant region or functional fragment thereof, IgD heavy chain constant region or functional fragment thereof, and IgE heavy chain constant region or functional fragment thereof.

[0136] In some embodiments, the first binding domain comprises a first immunoglobulin constant region (Fc region). In some embodiments, the second binding domain comprises asecond Fc region. In some embodiments, the first Fc region, the second Fc region, or a combination thereof is selected from the group consisting of an IgGl Fc region or a functional fragment thereof, an IgG2 Fc region or a functional fragment thereof, an IgG3 Fc region or a functional fragment thereof, an IgGAl Fc region or a functional fragment thereof, an IgGA2 Fc region or a functional fragment thereof, an IgG4 Fc region or a functional fragment thereof, an IgJ Fc region or a functional fragment thereof, an IgM Fc region or a functional fragment thereof, an IgD Fc region or a functional fragment thereof, and an IgE Fc region or a functional fragment thereof.

[0137] In some embodiments, the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, a knob and hole bispecific IgG, a Fc-Fab, or a knob and hole bispecific Fc-Fab.

[0138] In some embodiments, the first binding domain further comprises: (a) a first antigen binding domain; (b) a first polypeptide; and (c) a second polypeptide, wherein the first polypeptide and the second polypeptide are non-contiguous. In some embodiments, the first polypeptide comprises a Light Chain Constant Region (CL); and the second polypeptide comprises a Heavy Chain Constant Region (CH).

[0139] In some embodiments, the second binding domain further comprises: (a) a second antigen binding domain; (b) a third polypeptide; and (c) a fourth polypeptide, wherein the third polypeptide and the fourth polypeptide are non-contiguous. In some embodiments, the third polypeptide comprises a Light Chain Constant Region (CL); and the fourth polypeptide comprises a Heavy Chain Constant Region (CH).

[0140] In some embodiments, the VH of the first antigen binding domain comprises a dimerization domain; the VL of the first antigen binding domain comprises a dimerization domain; the VH of the second antigen binding domain comprises a dimerization domain; the VL of the second antigen binding domain comprises a dimerization domain; the CH of the second polypeptide comprises a dimerization domain; the CL of the first polypeptide comprises a dimerization domain; the CH of the fourth polypeptide comprises a dimerization domain; the CL of the third polypeptide comprises a dimerization domain; or a combination thereof. In some embodiments, the VH and VL of the first antigen binding domain are dimerized; the VH and VL of the second antigen binding domain are dimerized; the CH of the fourth polypeptide and the CL of the third polypeptide are dimerized; the CH of the second polypeptide and CL of the first polypeptide are dimerized; the CH of the second polypeptide and the CH of the fourth polypeptide are dimerized, or a combination thereof, wherein any one of (a)-(f) arelinked through the dimerization domain. In some embodiments, the dimerization domain comprises a disulfide bond.

[0141] In some embodiments, “complementarity-determining region” or “CDR” can refer to variable regions of either H (heavy) or L (light) chains (e.g., VH and VL, respectively) and can contain the amino acid sequences capable of specifically binding to antigenic targets. For example, the CDR regions can account for the basic specificity of the antibody for a particular antigenic determinant structure. Such regions are also referred to as “hypervariable regions.” The CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains. The variable heavy and light chains of all canonical antibodies each have three CDR regions, each non-contiguous with the others (termed LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3) for the respective light (L) and heavy (H) chains. In some embodiments, nanobodies can comprise a single amino acid chain that can be considered to comprise four “framework sequences or regions” or FRs and three complementarity-determining regions” or CDRs. The nanobodies have three CDR regions, each non-contiguous with the others (termed CDR1, CDR2, CDR3). The delineation of the FR and CDR sequences is based on the IMGT unique numbering system for V-domains and V- like domains.

[0142] In some embodiments, the binding agent comprises a bispecific, or a multi-specific binding agent. In some embodiments, the binding agent comprises an antibody. In some embodiments, the binding agent comprises a bispecific antibody (e.g., EpiTACs). In some embodiments, the binding agent comprises a multispecific antibody. In some embodiments, the binding agent comprises an agonist and / or activating antibody. In some embodiments, the binding agent comprises an antagonist and / or inhibitory antibody. In some embodiments, the binding agent comprises a multispecific antibody, a bispecific diabody, a bispecific Fab2, bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, an immunocytokine, an IgG-cytokine, a knob and hole bispecific IgG, a Fc-Fab, a Fc-cytokine, a scFv-Fc-cytokine, a Fab-cytokine, or a knob and hole bispecific Fc-Fab. In some embodiments, the antibody is not an FcyR activating antibody, or a pro-inflammatory response antibody.Antibody-like Frameworks or Scaffolds

[0143] A wide variety of antibody / immunoglobulin frameworks or scaffolds can be employed in the binding agents as described herein, or multifunctional formats thereof, so long as the resulting polypeptide includes at least one binding region which specifically binds to thetarget antigen, e.g., a tumor antigen, among others. Such frameworks or scaffolds include the 5 main idiotypes of human immunoglobulins, or fragments thereof, and include immunoglobulins of other animal species, preferably having humanized aspects. Novel frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art.

[0144] In some embodiments, the binding agents as described herein, or multifunctional formats thereof, include non-immunoglobulin based antibodies using non-immunoglobulin scaffolds onto which CDRs can be grafted. Any non-immunoglobulin frameworks and scaffolds may be employed, as long as they comprise a binding region specific for the target antigen. Exemplary non-immunoglobulin frameworks or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA, and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc., Mountain View, CA), Protein A (Affibody AG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

[0145] Fibronectin scaffolds are typically based on fibronectin type III domain (e.g., the tenth module of the fibronectin type III (10 Fn3 domain)). The fibronectin type III domain has 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (see US 6,818,418). Because of this structure, the non-immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in a loop randomization and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo. These fibronectin-based molecules can be used as scaffolds where the loop regions of the molecule can be replaced with CDRs of the invention using standard cloning techniques.

[0146] The ankyrin technology is based on using proteins with ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets. The ankyrin repeat module typically is a about 33 amino acid polypeptide consisting of two anti-parallel a-helices and a P-turn. Binding of the variable regions can be optimized by using ribosome display.

[0147] Avimers are used by nature for protein-protein interactions and in human over 250 proteins are structurally based on A-domains. Avimers consist of a number of different “A- domain” monomers (2-10) linked via amino acid linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, U.S. Patent Application Publication Nos. 20040175756; 20050053973; 20050048512; and 20060008844.

[0148] Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with a large number of ligand variants (See e.g., US 5,831,012). Affibody molecules mimic antibodies, they have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. In spite of its small size, the binding site of affibody molecules is similar to that of an antibody.

[0149] Anticalins are known commercially, e.g., Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body liquids. The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast with antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acid residues, being just marginally bigger than a single immunoglobulin domain. The set of four loops, which makes up the binding pocket, shows pronounced structural plasticity and tolerates a variety of side chains. The binding site can thus be reshaped in a proprietary process in order to recognize prescribed target molecules of different shape with high affinity and specificity. One protein of lipocalin family, the bilin-binding protein (BBP) of Pieris Brassicae has been used to develop anticalins by mutagenizing the set of four loops. One example of a patent application describing anticalins is in PCT Publication No. WO 199916873.

[0150] Affilin molecules are small non-immunoglobulin proteins which are designed for specific affinities towards proteins and small molecules. New affilin molecules can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein. Affilin molecules do not show any structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are employed, one of which is gamma crystalline, a human structural eye lens protein and the other is “ubiquitin” superfamily proteins. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturing agents. This high stability is mainly due to the expanded beta sheet structure ofthe proteins. Examples of gamma crystalline derived proteins are described in W0200104144 and examples of “ubiquitin-like” proteins are described in W02004106368.

[0151] Protein epitope mimetics (PEM) are medium-sized, cyclic, peptide-like molecules (MW l-2kDa) mimicking beta-hairpin secondary structures of proteins, the major secondary structure involved in protein-protein interactions.

[0152] Domain antibodies (dAbs) can be used in the binding agents as disclosed herein or multifunctional formats thereof. Domain antibodies (dAbs) can be small functional binding fragments of antibodies, corresponding to the variable regions of either the heavy or light chains of antibodies. Domain antibodies are well expressed in bacterial, yeast, and mammalian cell systems. Further details of domain antibodies and methods of production thereof are known in the art (see, for example, U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; European Patents 0368684 & 0616640; WO05 / 035572, W004 / 101790, W004 / 081026, W004 / 058821, W004 / 003019 and W003 / 002609. Nanobodies are derived from the heavy chains of an antibody.

[0153] A nanobody typically comprises a single variable domain and two constant domains (CH2 and CH3) and retains antigen-binding capacity of the original antibody. Nanobodies can be prepared by methods known in the art (See e.g., U.S. Pat. No. 6,765,087, U.S. Pat. No. 6,838,254, WO 06 / 079372). Unibodies consist of one light chain and one heavy chain of an IgG4 antibody. Unibodies may be made by the removal of the hinge region of IgG4 antibodies. Further details of unibodies and methods of preparing them may be found in W02007 / 059782. Effector function and Fc variants

[0154] In some embodiments, binding agents as disclosed herein comprises an Fc region, e.g., as described herein. In some embodiments, the Fc region is a wildtype Fc region, e.g., a wildtype human Fc region. In some embodiments, the Fc region comprises a variant, e.g., an Fc region comprising an addition, substitution, or deletion of at least one amino acid residue in the Fc region which results in, e.g., reduced or ablated affinity for at least one Fc receptor.

[0155] The Fc region of an antibody interacts with a number of receptors or ligands including Fc Receptors (e.g., FcyRI, FcyRIIA, FcyRIIIA), the complement protein Clq, and other molecules such as proteins A and G. These interactions are essential for a variety of effector functions and downstream signaling events including: antibody dependent cell- mediated cytotoxicity (ADCC), Antibody-dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDC).

[0156] In some embodiments, binding agents as disclosed herein comprise a variant Fc region having reduced, e.g., ablated, affinity for an Fc receptor, e.g., an Fc receptor describedherein. In some embodiments, the reduced affinity is compared to an otherwise similar antibody with a wildtype Fc region.

[0157] In some embodiments, binding agents as disclosed herein comprise a variant Fc region having one or more of the following properties: (1) reduced effector function (e.g., reduced ADCC, ADCP and / or CDC); (2) reduced binding to one or more Fc receptors; and / or (3) reduced binding to Clq complement. In some embodiments, the reduction in any one, or all of properties ( 1 )-(3 ) is compared to an otherwise similar antibody with a wildtype Fc region.

[0158] In some embodiments, binding agents as disclosed herein comprise a variant Fc region having reduced affinity to a human Fc receptor, e.g., FcyR I, FcyR II and / or FcyR III. In some embodiments, binding agents as disclosed herein comprise a variant Fc region having a human IgGl region or a human IgG4 region.

[0159] Exemplary Fc region variants are disclosed in Saunders O, (2019) Frontiers in Immunology; vol 10, articlel296, the entire contents of which is hereby incorporated by reference. In some embodiments, binding agents as disclosed herein comprise a pro-body. In some embodiments, the binding agents disclosed herein comprise a pro-body. A pro-body, such as a "masked" antibody or molecule, can refer to a modified form of an antibody or therapeutic protein that is designed to remain inactive until it encounters a specific target in the body. For example, in a pro-body, the active binding region of the molecule is concealed or masked by an additional component, such as a peptide or a chemical linker. When the pro-body encounters its specific target, such as a specific enzyme or marker expressed on cancer cells, the masking component is selectively cleaved or modified. Once the masking component is removed, the active binding region of the pro-body is exposed, allowing can bind specifically to its target. Multifunctional binding agents

[0160] As used herein, a “multifunctional” or a “multispecific” binding agent refers to binding agents, e.g., a polypeptide, that has two or more functionalities, e.g., two or more binding specificities. In some embodiments, the functionalities can include one or more immune cell engagers, one or more tumor binding molecules, and other moieties described herein. In some embodiments, binding agents disclosed herein comprise a multispecific binding agent. In some embodiments, the multispecific binding agent is a multispecific antibody binding agent, e.g., a bispecific antibody binding agent. In some embodiments, the multifunctional binding agents further includes a tumor antigen moiety. In some embodiments, the tumor-targeting moiety is an antigen, e.g., a cancer antigen. In some embodiments, the cancer antigen is a tumor antigen.

[0161] In some embodiments, an antibody binding agent is a multispecific or multifunctional antibody binding agent, e.g., it comprises a plurality of immunoglobulin variable domains sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In some embodiments, a multispecific antibody binding agent comprises a third, fourth or fifth immunoglobulin variable domain. In some embodiments, a multispecific antibody binding agent is a bispecific antibody binding agent, a trispecific antibody binding agent, or a tetraspecific antibody binding agent.

[0162] Cancer” as used herein can encompass all types of oncogenic processes and / or cancerous growths. In embodiments, cancer includes primary tumors as well as metastatic tissues or malignantly transformed cells, tissues, or organs. In embodiments, cancer encompasses all histopathologies and stages, e.g., stages of invasiveness / severity, of a cancer. In embodiments, cancer includes relapsed and / or resistant cancer. The terms “cancer” and “tumor” can be used interchangeably. For example, both terms encompass solid and liquid tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors.

[0163] In some embodiments, the multifunctional or multispecific (e.g., bi-, tri-, tetra- specific) binding agents as disclosed herein further include, e.g., are engineered to further contain, one or more tumor specific targeting moieties that direct the binding agent to a tumor cell.

[0164] In certain embodiments, the multifunctional or multispecific binding agents as disclosed herein further include a tumor-targeting moiety. The tumor targeting moiety can be chosen from an antibody binding agent (e.g., an antigen binding domain as described herein), a receptor or a receptor fragment, or a ligand or a ligand fragment, or a combination thereof. In some embodiments, the tumor targeting moiety associates with, e.g., binds to, a tumor cell (e.g., a molecule, e.g., antigen, present on the surface of the tumor cell). In certain embodiments, the tumor targeting moiety targets, e.g., directs the multifunctional or multispecific binding agents as disclosed herein to a cancer (e.g., a cancer or tumor cells). In some embodiments, the cancer is chosen from a solid cancer, a metastatic cancer, or a combination thereof.

[0165] In some embodiments, the multifunctional or multispecific binding agent, e.g., the tumor-targeting moiety, binds to a solid tumor antigen or a stromal antigen. The solid tumor antigen can be present on a solid tumor, or a metastatic lesion thereof. In some embodiments, the solid tumor is non-small cell lung cancer (NSCLC). For example, the solid tumor antigen can be present on a tumor, e.g., a tumor of a class typified by having one or more of: limited tumor perfusion, or compressed blood vessels.

[0166] In some embodiments, the antibody binding agent binds to a cancer antigen, e.g., a tumor antigen or a stromal antigen. In some embodiments, the cancer antigen is, e.g., a mammalian, e.g., a human, cancer antigen. For example, the antibody binding agent binds specifically to an epitope, e.g., linear or conformational epitope, on the cancer antigen.

[0167] In some embodiments, a multispecific antibody binding agent is a bispecific antibody binding agent. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody binding agent is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody binding agent comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In some embodiments, a bispecific antibody binding agent comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In some embodiments, a bispecific antibody binding agent comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In some embodiments, a bispecific antibody binding agent comprises a scFv or a Fab, or fragment thereof, have binding specificity for a first epitope and a scFv or a Fab, or fragment thereof, have binding specificity for a second epitope.

[0168] In some embodiments, an antibody binding agent comprises a diabody, and a singlechain molecule, as well as an antigen-binding fragment of an antibody (e.g., Fab, F(ab’)2, and Fv). For example, an antibody binding agent can include a heavy (H) chain variable domainsequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, an antibody binding agent comprises or consists of a heavy chain and a light chain (referred to herein as a half antibody. In another example, an antibody binding agent includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab’, F(ab’)2, Fc, Fd, Fd’, Fv, single chain antibodies (scFv for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen or receptor. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgGl, IgG2, IgG3, and IgG4) of antibodies. The preparation of antibody binding agents can be monoclonal or polyclonal. An antibody binding agent can also be a human, humanized, CDR-grafted, or in vitro generated antibody. The antibody can have a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4. The antibody can also have a light chain chosen from, e.g., kappa or lambda. The term “immunoglobulin” (Ig) is used interchangeably with the term “antibody” herein.

[0169] Examples of antigen-binding fragments of an antibody binding agent include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv), see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0170] Antibody binding agents include intact binding agents as well as functional fragments thereof. Constant regions of the antibody binding agents can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).

[0171] Antibody binding agents can also be single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. According to another aspect of the invention, a single domain antibody is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678, for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention.

[0172] The VH and VL regions can be subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR), interspersed with regions that are more conserved, termed “framework regions” (FR or FW).

[0173] The extent of the framework region and CDRs has been precisely defined by a number of methods (see, 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. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

[0174] The terms “complementarity determining region,” and “CDR,” as used herein refer to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3).

[0175] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of known schemes, including those described by Kabat et al. (1991),“Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme). As used herein, the CDRs defined according the “Chothia” number scheme are also sometimes referred to as “hypervariable loops.”

[0176] For example, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).

[0177] Each VH and VL typically includes three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0178] The antibody binding agent can be a polyclonal or a monoclonal antibody.

[0179] The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody binding agents of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. A monoclonal antibody can be made by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0180] The antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods, or by yeast display.

[0181] Phage display and combinatorial methods for generating antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92 / 18619; Dower et al. International Publication No. WO 91 / 17271; Winter et al. International Publication WO 92 / 20791; Markland et al. International Publication No. WO 92 / 15679; Breitling et al. International Publication WO 93 / 01288; McCafferty et al. International Publication No. WO 92 / 01047; Garrard et al. International Publication No. WO 92 / 09690; Ladner et al. International Publication No. WO 90 / 02809; Fuchs et al. (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246: 1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al.(1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).

[0182] The yeast display method for generating or identifying antibodies is known in the art, e.g., as described in Chao et al. (2006) Nature Protocols l(2):755-68, the entire contents of which is incorporated by reference herein.

[0183] In some embodiments, the antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods of producing rodent antibodies are known in the art.

[0184] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91 / 00906, Kucherlapati et al. PCT publication WO 91 / 10741; Lonberg et al. International Application WO 92 / 03918; Kay et al. International Application 92 / 03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L.L. et al. 1994 Nature Genet. 7: 13-21; Morrison, S.L. et al. 1994 Proc. Natl. Acad. Sci. USA 81 :6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21 : 1323-1326).

[0185] An antibody binding agent can be one in which the variable region, or a portion thereof, e.g., the CDRs, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibody binding agents generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention.

[0186] An “effectively human” protein is a protein that does substantially not evoke a neutralizing antibody response, e.g., the human anti-murine antibody (HAMA) response. HAMA can be problematic in a number of circumstances, e.g., if the antibody binding agent is administered repeatedly, e.g., in treatment of a chronic or recurrent disease condition. A HAMA response can make repeated antibody administration potentially ineffective because of an increased antibody clearance from the serum (see, e.g., Saleh et al., Cancer Immunol. Immunother., 32: 180-190 (1990)) and also because of potential allergic reactions (see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0187] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Patent Publication PCT / US86 / 02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240: 1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80: 1553-1559).

[0188] A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDRs (of heavy and or light immunoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding to the antigen. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDRs is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.” In some embodiments, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

[0189] As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

[0190] An antibody binding agent can be humanized by methods known in the art (see e.g., Morrison, S. L., 1985, Science 229: 1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762, the contents of all of which are hereby incorporated by reference).

[0191] Humanized or CDR-grafted antibody binding agents can be produced by CDR- grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced. See e.g., U.S. Patent 5,225,539; Jones et al. 1986 Nature 321 :552-525; Verhoeyanet al. 1988 Science 239: 1534; Beidler et al. 1988 J. Immunol. 141 :4053-4060; Winter US 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on March 26, 1987; Winter US 5,225,539), the contents of which is expressly incorporated by reference.

[0192] Also within the scope of the invention are humanized antibody binding agents in which specific amino acids have been substituted, deleted or added. Criteria for selecting amino acids from the donor are described in US 5,585,089, e.g., columns 12-16 of US 5,585,089, e.g., columns 12-16 of US 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 Al, published on December 23, 1992.

[0193] The antibody binding agent can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target protein.

[0194] In yet other embodiments, the antibody binding agent has a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgGl, IgG2, IgG3, and IgG4. In another embodiment, the antibody binding agent has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and / or complement function). In some embodiments the antibody has: effector function; and can fix complement. In other embodiments the antibody does not; recruit effector cells; or fix complement. In another embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[0195] Methods for altering an antibody constant region are known in the art. Antibodies with altered function, e.g. altered affinity for an effector ligand, such as FcR on a cell, or the Cl component of complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388,151 Al, U.S. Pat.No. 5,624,821 and U.S. Pat. No. 5,648,260, the contents of all of which are hereby incorporated by reference). Similar type of alterations could be described which if applied to the murine, or other species immunoglobulin would reduce or eliminate these functions.

[0196] An antibody binding agent can be derivatized or linked to another functional binding agent (e.g., another peptide or protein). As used herein, a “derivatized” antibody binding agent is one that has been modified. Methods of derivatization include but are not limited to the addition of a fluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinity ligand such as biotin. Accordingly, the antibody binding agents of the invention are intended to include derivatized and otherwise modified forms of the antibodies disclosed herein, including immunoadhesion molecules. For example, an antibody binding agent can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0197] One type of derivatized antibody binding agent is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxy succinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.CDR-grafted scaffolds

[0198] In some embodiments, the antibody binding agent is a CDR-grafted scaffold domain. In some embodiments, the scaffold domain is based on a fibronectin domain, e.g., fibronectin type III domain. The overall fold of the fibronectin type III (Fn3) domain is closely related to that of the smallest functional antibody fragment, the variable domain of the antibody heavy chain. There are three loops at the end of Fn3; the positions of BC, DE and FG loops approximately correspond to those of CDR1, 2 and 3 of the VH domain of an antibody. Fn3 does not have disulfide bonds; and therefore Fn3 is stable under reducing conditions, unlike antibodies and their fragments (see, e.g., WO 98 / 56915; WO 01 / 64942; WO 00 / 34784). An Fn3 domain can be modified (e.g., using CDRs or hypervariable loops described herein) or varied, e.g., to select domains that bind to an antigen / marker / cell described herein.

[0199] In some embodiments, a scaffold domain, e.g., a folded domain, is based on an antibody, e.g., a “minibody” scaffold created by deleting three beta strands from a heavy chainvariable domain of a monoclonal antibody (see, e.g., Tramontane et al., 1994, J Mol. Recognit. 7:9; and Martin et al., 1994, EMBO J. 13:5303-5309). The “minibody” can be used to present two hypervariable loops. In some embodiments, the scaffold domain is a V-like domain (see, e.g., Coia et al. WO 99 / 45110) or a domain derived from tendami statin, which is a 74 residue, six-strand beta sheet sandwich held together by two disulfide bonds (see, e.g., McConnell and Hoess, 1995, J Mol. Biol. 250:460). For example, the loops of tendamistatin can be modified (e.g., using CDRs or hypervariable loops) or varied, e.g., to select domains that bind to a marker / antigen / cell described herein. Another exemplary scaffold domain is a beta-sandwich structure derived from the extracellular domain of CTLA-4 (see, e.g., WO 00 / 60070).

[0200] Other exemplary scaffold domains include but are not limited to T-cell receptors; MHC proteins; extracellular domains (e.g., fibronectin Type III repeats, EGF repeats); protease inhibitors (e.g., Kunitz domains, ecotin, BPTI, and so forth); TPR repeats; trifoil structures; zinc finger domains; DNA-binding proteins; particularly monomeric DNA binding proteins; RNA binding proteins; enzymes, e.g., proteases (particularly inactivated proteases), RNase; chaperones, e.g., thioredoxin, and heat shock proteins; and intracellular signaling domains (such as SH2 and SH3 domains). See, e.g., US 20040009530 and US 7,501,121, incorporated herein by reference.

[0201] In some embodiments, a scaffold domain is evaluated and chosen, e.g., by one or more of the following criteria: (1) amino acid sequence, (2) sequences of several homologous domains, (3) 3-dimensional structure, and / or (4) stability data over a range of pH, temperature, salinity, organic solvent, oxidant concentration. In some embodiments, the scaffold domain is a small, stable protein domain, e.g., a protein of less than 100, 70, 50, 40 or 30 amino acids. The domain may include one or more disulfide bonds or may chelate a metal, e.g., zinc.Antibody-Based Fusions

[0202] A variety of formats can be generated which contain additional binding entities attached to the N or C terminus of antibodies. These fusions with single chain or disulfide stabilized Fvs or Fabs result in the generation of tetravalent binding agents with bivalent binding specificity for each antigen. Combinations of scFvs and scFabs with IgGs enable the production of binding agents which can recognize three or more different antigens.Antibody-Fab Fusion

[0203] Antibody-Fab fusions are bispecific antibodies comprising a traditional antibody to a first target and a Fab to a second target fused to the C terminus of the antibody heavy chain. Commonly the antibody and the Fab will have a common light chain. Antibody fusions can be produced by (1) engineering the DNA sequence of the target fusion, and (2) transfecting thetarget DNA into a suitable host cell to express the fusion protein. It seems like the antibody- scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and theN-terminus of the scFv, as described by Coloma, J. et al. (1997) Nature Biotech 15: 159. Antibody-scFv Fusion

[0204] Antibody-scFv Fusions are bispecific antibodies comprising a traditional antibody and a scFv of unique specificity fused to the C terminus of the antibody heavy chain. The scFv can be fused to the C terminus through the Heavy Chain of the scFv either directly or through a linker peptide. Antibody fusions can be produced by (1) engineering the DNA sequence of the target fusion, and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. It seems like the antibody-scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv, as described by Coloma, J. et al. (1997) Nature Biotech 15: 159.Variable Domain Immunoglobulin DVD

[0205] A related format is the dual variable domain immunoglobulin (DVD), which are composed of VH and VL domains of a second specificity place upon the N termini of the V domains by shorter linker sequences.

[0206] Other exemplary multispecific antibody formats include, e.g., those described in the following US20160114057A1, US20130243775A1, US20140051833, US20130022601,US20150017187A1, US20120201746A1, US20150133638A1, US20130266568A1US20160145340A1, WO2015127158A1, US20150203591A1, US20140322221A1US20130303396A1, US20110293613, US20130017200A1, US20160102135A1WO2015197598A2, WO2015197582A1, US9359437, US20150018529, WO2016115274A1, WO20 16087416A1, US20080069820A1, US9145588B, US7919257, andUS20150232560A1. Exemplary multispecific binding agents utilizing a full antibody- Fab / scFab format include those described in the following, US9382323B2, US20140072581A1, US20140308285A1, US20130165638A1, US20130267686A1,US20140377269A1, US7741446B2, and WO 1995009917A1. Exemplary multispecific binding agents utilizing a domain exchange format include those described in the following, US20150315296A1, W02016087650A1, US20160075785A1, WO2016016299A1,US20160130347A1, US20150166670, US8703132B2, US20100316645, US8227577B2, US20130078249.Fc-containing multifunctional or multispecific binding agents

[0207] In some embodiments, the multifunctional or multispecific binding agents as disclosed herein includes an immunoglobulin constant region (e.g., an Fc region). ExemplaryFc regions can be chosen from the heavy chain constant regions of IgGl, IgG2, IgG3 or IgG4; more particularly, the heavy chain constant region of human IgGl, IgG2, IgG3, or IgG4.

[0208] In some embodiments, the immunoglobulin chain constant region (e.g., the Fc region) is altered, e.g., mutated, to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function.

[0209] In other embodiments, an interface of a first and second immunoglobulin chain constant regions (e.g., a first and a second Fc region) is altered, e.g., mutated, to increase or decrease dimerization, e.g., relative to a non-engineered interface, e.g., a naturally-occurring interface. For example, dimerization of the immunoglobulin chain constant region (e.g., the Fc region) can be enhanced by providing an Fc interface of a first and a second Fc region with one or more of: a paired protuberance-cavity (“knob-in-a hole”), an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer to homomultimer forms, e.g., relative to a non-engineered interface.

[0210] In some embodiments, the multifunctional or multispecific binding agents include a paired amino acid substitution at a position chosen from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, e.g., of the Fc region of human IgGl For example, the immunoglobulin chain constant region (e.g., Fc region) can include a paired an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole), and T366W (e.g., corresponding to a protuberance or knob).

[0211] In other embodiments, the multifunctional binding agent includes a half-life extender, e.g., a human serum albumin or an antibody binding agent to human serum albumin.

[0212] In some embodiments, the binding agent comprises an antibody having a heavy chain (HC) sequence, a variable heavy (VH) sequence, a light chain (LC) sequence, and a variable light (VL) sequence. In some embodiments, the binding agent comprises an antibody having an HC sequence and a VH sequence. In some embodiments, the binding agent comprises an antibody having an HC sequence, a VH sequence, an LC sequence, and / or a VL sequence listed in Table 1 or Table 2. In some cases, the binding agent comprises an antibody, wherein the antibody binds the same epitope as any one of the antibodies listed in Table 1 orTable 2

[0213] In some embodiments, the binding agent comprises an antibody having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or more sequence identity to any one of SEQ ID NOs: 1-353. In some cases, the binding agent comprises an antibody having at most about 60 %, at most about 65 %, at most about 70 %, at most about 75 %, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, or at most about 99.9% sequence identity to any one of SEQ ID NOs: 1-353. In some cases, the binding agent comprises an antibody having 100 % sequence identity to any one of SEQ ID NOs: 1-353 In some cases, the binding agent comprises an antibody having a sequence that has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25 or more amino acid substitutions or mutations relative to any one of SEQ ID NOs: 1-353. In some cases, the binding agent comprises an antibody having a sequence that has at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 10, at most about 11, at most about 12, at most about 13, at most about 14, at most about 15, at most about 16, at most about 17, at most about 18, at most about 19, at most about 20, at most about 21, at most about 22, at most about 23, at most about 24, or at most about 25 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 1-353. Table 1 below lists the sequences of SEQ ID NOs: 1-353.Table 1. Exemplary antibody sequences of the binding agents disclosed herein.

[0214] The sequences listed in Table 1 (SEQ ID NOs: 1-353) are amino acid molecules. The sequences listed in Table 1 (SEQ ID NOs: 1-353) are amino acid molecules that are synthetic constructs. The sequences listed in Table 1 (SEQ ID NOs: 1-353) for HC sequences (heavy chain), VH sequence (variable heavy chain sequence), LC sequences (light chain), VL sequence (variable light chain sequence) are amino acid molecules that are synthetic constructs.

[0215] In some embodiments, the binding agent comprises an antibody having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or more sequence identity to any one of SEQ ID NOs: 354- 723. In some cases, the binding agent comprises an antibody having at most about 60 %, at most about 65 %, at most about 70 %, at most about 75 %, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, or at most about 99.9% sequence identity to any one of SEQ ID NOs: 354- 723. In some cases, the binding agent comprises an antibody having 100 % sequence identity to any one of SEQ ID NOs: 354-723. In some cases, the binding agent comprises an antibody having a sequence that has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25 or more amino acid substitutions or mutations relative to any one of SEQ ID NOs: 354-723. In some cases, the binding agent comprises an antibody having a sequence that has at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 10, at most about 11, at most about 12, at most about 13, at most about 14, at most about 15, at most about 16, at most about 17, at most about 18, at most about 19, at most about 20, atmost about 21, at most about 22, at most about 23, at most about 24, or at most about 25 amino acid substitutions or mutations relative to any one of SEQ ID NOs: 354-723. Table 2 below lists the sequences of SEQ ID NOs: 354-723.Table 2. Additional exemplary antibody sequences of the binding agents disclosed herein.

[0216] The sequences listed in Table 2 (SEQ ID NOs: 354-633; 684-723) are amino acid molecules. The sequences listed in Table 2 (SEQ ID NOs: 354-633; 684-723) are amino acid molecules that are synthetic constructs. The sequences listed in Table 2 (SEQ ID NOs: 354- 633; 684-723) for HC sequences (heavy chain), VH sequence (variable heavy chain sequence), LC sequences (light chain), VL sequence (variable light chain sequence) are amino acid molecules that are synthetic constructs.

[0217] In other embodiments, the binding agent comprises an antibody, wherein the antibody comprises an antigen-binding domain (Fab) from any antigen-binding molecule, such as an antigen-binding domain from a clinically approved antibody. Some exemplary therapeutic monoclonal antibodies approved or in review in the EU or US are provided in Table 3 below.Table 3. Exemplary therapeutic monoclonal antibodiesFirst Binding Domain

[0218] In some embodiments, the binding agent(s) of the present disclosure comprises a first binding domain. In some embodiments, the first binding domain binds to an epitope of the membrane-bound target protein(s) disclosed herein. In some cases, the first binding domain comprises an antigen binding (Fab) domain of an antibody. In some cases, the antibody or the antigen binding (Fab) domain binds the same epitope as any one of the antibodies listed in Table 1 or Table 2. In some embodiments, the first binding domain can refer to an arm of an antibody. In some embodiments, the first binding domain can refer to a target binding arm of an antibody. In some embodiments, the first binding domain can refer to a target binding arm of a bispecific antibody.

[0219] In some embodiments, the first binding domain comprises a Fab domain. In some cases, the first binding domain comprises a Fab domain, wherein the Fab domain can be from any antigen-binding molecule, such as an antigen-binding domain from a clinically approved antibody (e.g., an antibody from Table 3). In some embodiments, the first binding domain comprises a VL and / or VH domain.

[0220] In some embodiments, the first binding domain comprises a VL domain. In some embodiments, the first binding domain comprises a VL domain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or more sequence identity to any one of the VL sequences listed in Table 1. In some cases, the first binding domain comprises a VL domain having at most about 60 %, at most about 65 %, at most about 70 %, at most about 75 %, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, or at most about 99.9% sequence identity to any one of the VL sequences listed in Table 1. In some cases, the first binding domain comprises a VL domain having a sequence that has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25 or more amino acid substitutions or mutations relative to any one of the VL sequences listed in Table 1. In some cases, the first binding domain comprises a VL domain having a sequence that has at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 10, at most about 11, at most about 12, at most about 13, at most about 14, at most about 15, at most about 16, at most about 17, at most about 18, at most about 19, at most about 20, at most about 21, at most about 22, at most about 23, at most about 24, or at most about 25 amino acid substitutions or mutations relative to any one of the VL sequences listed in Table 1. In some embodiments, the first binding domain comprises a VL domain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or more sequence identity to any one of the VL sequences listed in Table 2. In some cases, the first binding domain comprises a VL domain having at most about 60 %, at most about 65 %, at most about 70 %, at most about 75 %, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, or at most about 99.9% sequence identity to any one of the VL sequences listed in Table 2. In some cases, the first binding domain comprises a VL domain having a sequence that has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25 or more amino acid substitutions or mutations relative toany one of the VL sequences listed in Table 2. In some cases, the first binding domain comprises a VL domain having a sequence that has at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 10, at most about 11, at most about 12, at most about 13, at most about 14, at most about 15, at most about 16, at most about 17, at most about 18, at most about 19, at most about 20, at most about 21, at most about 22, at most about 23, at most about 24, or at most about 25 amino acid substitutions or mutations relative to any one of the VL sequences listed in Table 2.

[0221] In some embodiments, the first binding domain comprises a VH domain. In some embodiments, the first binding domain comprises a VH domain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or more sequence identity to any one of the VH sequences listed in Table 1. In some cases, the first binding domain comprises a VH domain having at most about 60 %, at most about 65 %, at most about 70 %, at most about 75 %, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, or at most about 99.9% sequence identity to any one of the VH sequences listed in Table 1. In some cases, the first binding domain comprises a VH domain having a sequence that has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25 or more amino acid substitutions or mutations relative to any one of the VH sequences listed in Table 1. In some cases, the first binding domain comprises a VH domain having a sequence that has at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 10, at most about 11, at most about 12, at most about 13, at most about 14, at most about 15, at most about 16, at most about 17, at most about 18, at most about 19, at most about 20, at most about 21, at most about 22, at most about 23, at most about 24, or at most about 25 amino acid substitutions or mutations relative to any one of the VH sequences listed in Table 1. In some embodiments, the first binding domain comprises a VH domain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or more sequence identity to any one of the VH sequences listed in Table 2. In some cases, the first binding domain comprises a VH domainhaving at most about 60 %, at most about 65 %, at most about 70 %, at most about 75 %, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, or at most about 99.9% sequence identity to any one of the VH sequences listed in Table 2. In some cases, the first binding domain comprises a VH domain having a sequence that has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25 or more amino acid substitutions or mutations relative to any one of the VH sequences listed in Table 2. In some cases, the first binding domain comprises a VH domain having a sequence that has at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 10, at most about 11, at most about 12, at most about 13, at most about 14, at most about 15, at most about 16, at most about 17, at most about 18, at most about 19, at most about 20, at most about 21, at most about 22, at most about 23, at most about 24, or at most about 25 amino acid substitutions or mutations relative to any one of the VH sequences listed in Table 2.Second Binding Domain

[0222] In some embodiments, the binding agent(s) of the present disclosure comprises a second binding domain. In some cases, the second binding domain can specifically bind to at least one endogenous cell surface receptor. In some embodiments, the binding of the second binding domain to the at least one endogenous cell surface receptor results in the internalization of the endogenous cell surface receptor and the binding agent. In other embodiments, the endogenous cell surface receptor can be internalized on its own, and pull in the target protein, due to simultaneous binding of binding agent to the endogenous cell surface receptor and target protein. In certain embodiments, the endogenous cell surface receptor is membrane associated.

[0223] In some embodiments, the second binding domain of the binding agents provided herein can be a cytokine (e.g., a chemokine), or an isoform or a derivative capable of binding thereof. A functional derivative of a cytokine can be any agent that possesses the binding activity of a cytokine. For example, in some embodiments, the second binding domain of the bispecific binding agents can be an antagonistic variant of a cytokine which does not have a functional effect but binds to the endogenous cell surface receptors of the cytokine. In this instance, the antagonistic variant of a cytokine is a functional derivative of the cytokine. In other embodiments, the second binding domain of the bispecific binding agents can be abinding agent (e.g., an antibody or a fragment thereof, a peptide, or a small molecule) that binds to the endogenous cell surface receptors of a cytokine. A functional derivative of a cytokine can be any agent that maintains binding affinity and / or selectivity of the cytokine to a cytokine receptor. The functional derivative may or may not have the same activity as the native cytokine. For example, a functional derivative of a cytokine may share the binding affinity of the cytokine to the cytokine receptor but the functional derivative may lack the agonistic or antagonistic activity of the cytokine. In certain embodiments, a functional derivative of a cytokine shares the binding affinity of the cytokine to the cytokine receptor and the functional derivative maintains similar agonistic or antagonistic activity relative to the cytokine.

[0224] Cytokines are a diverse class of soluble extracellular proteins and can include interleukins, chemokines, interferons, tumor necrosis factors, prolactins, transforming growth factor betas, and lymphokines. Upon binding to their cognate receptors on the surface of cells, cytokines trigger downstream signaling, which is in many cases coupled to internalization of the cytokine-receptor complex. Cytokines exert their action through high-affinity receptors on the cell surface that are linked to pathways of cellular activation, survival, proliferation and differentiation. Cross-linking of receptor subunits on the outside of the cell membrane can lead to abutting of kinases associated with the intracellular receptor tails. This intracellular association of signaling molecules results in phosphorylation of tyrosine residues in the receptor tail and binding of further signaling molecules that have phospho-tyrosine-binding domains. In some instances, different activated receptor cytoplasmic domains can bind a common signaling molecule or family of signaling molecules. Hapel AJ and Stanley RE. Cytokines, Receptors and Signaling Pathways Involved in Macrophage and Dendritic Cell Development. In: Madame Curie Bioscience Database. Austin (TX): Landes Bioscience; 2000- 2013. Thus, the triggering event (such as the binding of a cytokine to its receptor) required to trigger a cytokine mediated signaling can be much lower than that is required for a noncytokine receptor. Cytokine is used as its common meaning in the field and refers to a broad category of peptides important in cell signaling. Some non-limiting examples of cytokines include chemokines, interferons, interleukins, prolactins, transforming growth factor betas, lymphokines, and tumor necrosis factors. Chemokines, or chemotactic cytokines, are small chemoattractant secreted molecules regulating cell positioning and cell recruitment into tissues, playing a pivotal role in embryogenesis, tissue development and immune response. Approximately 50 chemokines and 20 chemokine receptors have been discovered so far. Chemokines and their receptors have been reported to play important roles in immune cell migration and inflammation, as well as in tumor initiation, promotion, and progression.Marcuzzi E, et al. Chemokines and Chemokine Receptors: Orchestrating Tumor Metastasi zati on. Int J Mol Sci. 2018 Dec 27;20(l):96. Chemokines can be widely divided into two major groups based on their prominent functions: inflammatory and homeostatic chemokines. Among inflammatory chemokines, which are induced by inflammation, some non-limiting examples include CXCL1, CXCL2, CXCL3, CXCL5, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, and CXCL14. On the other hand, homeostatic chemokines such as, without being limited to, CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12 and CXCL13 are constitutively expressed and are involved in homeostatic leukocyte trafficking.

[0225] In some embodiments, the second binding domain comprises a cytokine, or a biologically active fragment thereof (e.g., functional derivative of a cytokine), selected from the group comprising CXCL12, CCL1, CCL2, CCL3, CCL3L1, CCL4, CC4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL11, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, vMIPII, and vCXCl. In some embodiments, the chemokine comprises a CXC chemokine, or an isoform or a derivative capable of binding thereof. In certain non-limiting exemplary embodiments, the chemokine can be CXCL12, CCL1, CCL2, CCL3, CCL3L1, CCL4, CC4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL11, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, vMIPII, U83, and vCXCl. In some embodiments, the chemokine includes CCL1, CCL2, CCL3, CCL3L1, CCL4, CC4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28. In some embodiments, the chemokine includes CXCL12, CXCL11, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, XCL2. In some embodiments the chemokine includes vMIPII, U83, or vCXCl.

[0226] In some embodiments, the second binding domain can refer to an arm of an antibody. In some embodiments, the second binding domain can refer to a degrader binding arm of an antibody. In some embodiments, the second binding domain can refer to a degrader binding arm of a bispecific antibody. In some embodiments, the second binding domain of the binding agent(s) of the present disclosure, comprises a second binding domain variable heavy chain and a second binding domain variable light chain. In some embodiments, the secondbinding domain comprises specific binding to one or more membrane-associated proteins. In some cases, the second binding domain comprises an antigen binding (Fab) domain of an antibody. In some cases, the antibody or the antigen binding (Fab) domain binds the same epitope as any one of the antibodies listed in Table 1 or Table 2.

[0227] In some embodiments, the second binding domain comprises a Fab domain. In some cases, the second binding domain comprises a Fab domain, wherein the Fab domain can be from any antigen-binding molecule, such as an antigen-binding domain from a clinically approved antibody (e.g., an antibody from Table 3). In some embodiments, the second binding domain comprises a VL and / or VH domain.

[0228] In some embodiments, the second binding domain comprises a VL domain. In some embodiments, the second binding domain comprises a VL domain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or more sequence identity to any one of the VL sequences listed in Table 1. In some cases, the second binding domain comprises a VL domain having at most about 60 %, at most about 65 %, at most about 70 %, at most about 75 %, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, or at most about 99.9% sequence identity to any one of the VL sequences listed in Table 1. In some cases, the second binding domain comprises a VL domain having a sequence that has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25 or more amino acid substitutions or mutations relative to any one of the VL sequences listed in Table 1. In some cases, the second binding domain comprises a VL domain having a sequence that has at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 10, at most about 11, at most about 12, at most about 13, at most about 14, at most about 15, at most about 16, at most about 17, at most about 18, at most about 19, at most about 20, at most about 21, at most about 22, at most about 23, at most about 24, or at most about 25 amino acid substitutions or mutations relative to any one of the VL sequences listed in Table 1. In some embodiments, the second binding domain comprises a VL domain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, at least 99.9%, or more sequence identity to any one of the VL sequences listed in Table 2. In some cases, the second binding domain comprises a VL domain having at most about 60 %, at most about 65 %, at most about 70 %, at most about 75 %, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, or at most about 99.9% sequence identity to any one of the VL sequences listed in Table 2. In some cases, the second binding domain comprises a VL domain having a sequence that has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25 or more amino acid substitutions or mutations relative to any one of the VL sequences listed in Table 2. In some cases, the second binding domain comprises a VL domain having a sequence that has at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 10, at most about 11, at most about 12, at most about 13, at most about 14, at most about 15, at most about 16, at most about 17, at most about 18, at most about 19, at most about 20, at most about 21, at most about 22, at most about 23, at most about 24, or at most about 25 amino acid substitutions or mutations relative to any one of the VL sequences listed in Table 2.

[0229] In some embodiments, the second binding domain comprises a VH domain. In some embodiments, the second binding domain comprises a VH domain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or more sequence identity to any one of the VH sequences listed in Table 1. In some cases, the second binding domain comprises a VH domain having at most about 60 %, at most about 65 %, at most about 70 %, at most about 75 %, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, or at most about 99.9% sequence identity to any one of the VH sequences listed in Table 1. In some cases, the second binding domain comprises a VH domain having a sequence that has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, atleast about 25 or more amino acid substitutions or mutations relative to any one of the VH sequences listed in Table 1. In some cases, the second binding domain comprises a VH domain having a sequence that has at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 10, at most about 11, at most about 12, at most about 13, at most about 14, at most about 15, at most about 16, at most about 17, at most about 18, at most about 19, at most about 20, at most about 21, at most about 22, at most about 23, at most about 24, or at most about 25 amino acid substitutions or mutations relative to any one of the VH sequences listed in Table 1. In some embodiments, the second binding domain comprises a VH domain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or more sequence identity to any one of the VH sequences listed in Table 2. In some cases, the second binding domain comprises a VH domain having at most about 60 %, at most about 65 %, at most about 70 %, at most about 75 %, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, or at most about 99.9% sequence identity to any one of the VH sequences listed in Table 2. In some cases, the second binding domain comprises a VH domain having a sequence that has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25 or more amino acid substitutions or mutations relative to any one of the VH sequences listed in Table 2. In some cases, the second binding domain comprises a VH domain having a sequence that has at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 10, at most about 11, at most about 12, at most about 13, at most about 14, at most about 15, at most about 16, at most about 17, at most about 18, at most about 19, at most about 20, at most about 21, at most about 22, at most about 23, at most about 24, or at most about 25 amino acid substitutions or mutations relative to any one of the VH sequences listed in Table 2.

[0230] In some embodiments, contacting of the second binding domain to the membrane- associated protein results in the internalization of the membrane-associated protein and the binding agent. In some embodiments, contacting of the first binding domain to the soluble target protein, and the subsequent or simultaneous contacting of the second binding domain to the membrane-associated protein, results in the internalization of the membrane-associatedprotein, the binding agent, and the soluble target protein, and wherein the soluble target protein is then degraded.2. Membrane-associated protein and / or ceil surface receptor

[0231] Provided herein is a method of degrading a target protein, the method comprising contacting the target protein and a membrane-associated protein with a binding agent, wherein the membrane-associated protein is associated with a target cell; and wherein the contacting of the target protein and the membrane-associated protein with the binding agent leads to degradation of the target protein. In some embodiments, the membrane-associated protein is recycled to the target cell surface following the internalization of the binding agent.

[0232] In some embodiments, the binding agents disclosed herein comprises a second binding domain that specifically binds to a membrane-associated protein. In some cases, the membrane-associated protein can be a cell surface receptor. In some cases, the binding agents provided herein can specifically bind to one or more cell surface receptors. In some embodiments, the second binding domain specifically binds to one cell surface receptor. In some embodiments, the second binding domain specifically binds to no more than two cell surface receptors. In some embodiments, the second binding domain specifically binds to two cell surface receptors. In some embodiments, the endogenous cell surface receptor can be a monomeric receptor. In some embodiments, the endogenous cell surface receptor can form a complex with other molecules (e.g., an integrin). The endogenous cell surface receptors can be recycling receptors. For example, a recycling receptor as used herein refers to an endogenous cell surface receptor that specifically binds to a ligand, e.g., a cytokine, a chemokine, a growth factor or an isoform or a derivative capable of binding thereof and leads to internalization and degradation of the endogenous cell surface receptor and the cell expressing the receptor. In some embodiments, the degradation can occur through delivery of the target protein discussed herein to a lysosome via either a targeting or a recycling receptor. In some embodiments, the membrane-associated protein comprises an endogenous internalizing receptor, a degrader receptor, or a membrane-associated ubiquitin E3 ligase. In some embodiments, the endogenous internalizing receptor comprises targeting receptors and / or recycling receptors.

[0233] In some embodiments, the binding of the second binding domain to the at least one endogenous cell surface receptor results in the internalization of the endogenous cell surface receptor and the binding agent. In some embodiments, the binding of the second binding domain to the at least one endogenous cell surface receptor results in the degradation of the target protein bound to the binding agent disclosed herein. In certain embodiments, the bindingof the second binding domain to the at least one endogenous cell surface receptor results in the degradation of the target protein bound to the binding agent disclosed herein, but not the binding agent.

[0234] In certain embodiments, the endogenous cell surface receptor is membrane associated. Membrane proteins represent about a third of the proteins in living organisms and many membrane proteins are known in the field. Based on their structure, membrane proteins can be largely categorized into three main types: (1) integral membrane protein (IMP), which is permanently anchored or part of the membrane, (2) peripheral membrane protein, which is temporarily attached to the lipid bilayer or to other integral proteins, and (3) lipid-anchored proteins. The most common type of IMP is the transmembrane protein (TM), which spans the entire biological membrane. The endogenous cell surface receptor of the present disclosure includes single-pass and multi-pass membrane proteins. Single-pass membrane proteins cross the membrane only once, while multi-pass membrane proteins weave in and out, crossing several times.

[0235] In some case, the internalizing receptors and / or E3 ligases can be selected by expression levels in one cell type relative to other cell types, where the internalizing receptors and / or E3 ligases are enrichment in the selected cell type and / or tissue type. In some case, rapid selection of tumor- and tissue-specific degraders, including transmembrane E3 ubiquitin ligases, chemokine / cytokine receptors, and tissue-enriched internalizing receptors was achieved using an internal database (e.g., EpiAtlas containing 270+ tumor- and tissue-specific degraders), see Example 2.

[0236] Some non-limiting membrane proteins encompassed herein include cytokine receptors, insulin receptors, cell adhesion proteins or cell adhesion molecules (CAMs), receptor proteins, glycophorin, rhodopsin, Band 3, CD36, glucose permease, ion channels and gates, gap junction proteins, G protein coupled receptors (e.g., beta-adrenergic receptor), and seipin. In some exemplary embodiments, CAMs can include integrins, cadherins, neural cell adhesion molecules (NCAMs), or selectins, etc. In some cases, the cell surface receptor and / or membrane-associated protein can be referred to as an internalizing protein. In some embodiments, the membrane associated internalizing protein is selected from CEACAM5, CEACAM6, HER3, MUC1, CD205, CD166, PRLR, SLC34A2, ITGB6, LRRC15, MUC16, SLC39A6, AXL, CD40, CD228, MUC5A, ITGB1, STn, KAAG1, DLK1, 5T4, SEZ6, ADAM9, I-Ag7, ENPP3, CD46, CD56, ROR1, GPR20, TM4SF1, B7-H4, ALPP, LY6E, CLDN18, LY6G6D, GPR56, CD71, LGR5, LY75, CD276 / B7-H3, MST1R, MSLN, EpCAM, TNFRSF10B, STEAP1, MELTF, TROP2, CDH17, RNF43, and RNF128. In someembodiments, the cell surface receptor and / or membrane-associated protein is selected from one or more of the following: LGR5, HER3, LY75, MST1R, MSLN, EpCAM, TNFRSF10B, CD71, KIT, FGFR4, HER2, EGFR, CD30, CCR8, 0X40, CD79B, Nectin-4, BCMA, integrin 07, FcyRIIb, IL1RAP, CD33, MUC1, ITGB6, CEACAM5, CDH17, CD20, CD22, CD19, BAFF-R, CD38, TROP2, B7-H3, MARCH-1, MARCH-8, MARCH-9, GRAIL, Tissue factor, FOLR1, CD45, and TFRC. In some embodiments, the membrane-associated protein is not FcyRI, FcyRIIa, or FcyRIII.Tissue and cell-type specific receptors and / or membrane-associated proteins

[0237] In some embodiments, the membrane-associated protein comprises a tissue-type specific protein. In some embodiments, the membrane-associated protein comprises a cell-type specific protein. In some embodiments, the membrane-associated protein is enriched on a regulatory T cell when compared to an effector T cell. In some embodiments, the membrane- associated protein is enriched on an effector T cell when compared to a regulatory T cell.Cytokine Receptors

[0238] In some embodiments, the cell surface receptor and / or membrane-associated protein comprises a cytokine receptor. A cytokine receptor can include single-pass and multipass membrane-associated receptors. For instance, the cytokine receptor can be a chemokine receptor. Chemokine receptors, which are seven transmembrane spanning proteins coupled to G-proteins, are similarly divided into subfamilies based on their cysteine residues pattern: CXC, CC, CX3C, where C stands for cysteine and X represents non- cysteine amino acids. It has been reported that there is a significant ligand promiscuity among certain chemokine receptors, as some chemokines can bind to and signal through several chemokine receptors, both canonical and atypical ones. In contrast, some chemokines (e.g., CXCL12) are more selective. Some non-limiting examples of chemokine receptor include CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 (or ACKR3), XCR1, XCR2, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, ACKR1, ACKR2, ACKR4, and ACKR5. In some embodiments, the chemokine receptor includes CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CCR11. In some embodiments, the chemokine receptor includes CXCR7, CXCR4, CXCR3, CXCR1, CXCR2, CXCR5, CXCR6, CX3CR1, XCR1, XCR2. In some embodiments, the chemokine receptor includes ACKR1, ACKR2, CXCR7, ACKR4. In some embodiments, the second binding domain of the bispecific binding agent provided herein specifically binds to CXCR4. In some embodiments, the second binding domain of the binding agent provided hereinspecifically binds to CXCR7. In certain embodiments, the second binding domain of the binding agent provided herein specifically binds to CXCR4 and CXCR7.

[0239] In some embodiments, the cytokine receptor can be an interleukin receptor. Interleukin receptors are members of the immunoglobulin superfamily receptors and are transmembrane proteins defined by their structural similarity to immunoglobulins. They often contain an amino-terminal extracellular domain that holds the characteristic immunoglobulin fold. Interluekin receptors are typically involved with cell adhesion and the interaction between T cells and antigen presenting cells. Some non-limiting examples of interleukin receptors include CD25, IL2RB, IL2RG, IL3RA, IL4R, IL13RA1, IL13RA2, IL5RA, IL6R, IL7R, IL8R, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL15RA, CD4, IL17RA, IL17RC, IL17RB, IL17RE, IL27RA, IL18R1, IL20RA, IL20RB, IL22RA1, IL21R, IL28RA, IL31RA, ST2, IL1RAP, CSF1R, IL1R1, IL1RL2, IL1R2.

[0240] In some embodiments, the cytokine receptor can be an interferon receptor. All receptors involved in interferon signal transduction are classified as class II helical cytokine receptors (hCRs) sharing homologous structural folds and basic structural elements with other proteins including tissue factor, and the receptors for IL- 10, IL-20 and IL-22. 4 In the extracellular region, all members of this class of hCR have tandem domains consisting of ~100 amino acids each housing a type III fibronectin (FBN-III) domain with topology analogous to the immunoglobulin constant domain. With the exception of IFNAR1, which has a four- domain architecture, all other IFN receptors consist of two FBN-III domains. Some nonlimiting examples of interferon receptors include IFNAR1, IFNAR2, IFNGR1, IFNGR2.

[0241] In some embodiments, the cytokine receptor can be a prolactin receptor. The prolactin receptor (PRLR) is a membrane-bound, type I cytokine receptor. Some nonlimiting examples of prolactin receptors include EPOR, GHR, PRLR, CSF3R, LEPR, CSF1R.

[0242] In some embodiments, the cytokine receptor can be a tumor necrosis factor (TNF) receptor. In their active form, the majority of TNF receptors form trimeric complexes in the plasma membrane. Most TNF receptors contain transmembrane domains (TMDs), although some can be cleaved into soluble forms (e.g. TNFR1), and some lack a TMD entirely (e.g. DcR3). TNF receptors are primarily involved in apoptosis and inflammation, but they can also take part in other signal transduction pathways, such as proliferation, survival, and differentiation. Some non-limiting examples of TNF receptors include TNFR1, TNFR2, DR4, DR5, DCR1, DCR2, DR3, LTBR, BAFFR, TACI, OPG, RANK, CD40, ED AR, DCR3, FAS, and CD27.Receptor tyrosine kinases (RTKs), such as Growth Factor Receptors

[0243] In some embodiments, the cell surface receptor and / or membrane-associated protein can be a receptor tyrosine kinase (RTK). For example, the RTK can include, but is not limited to, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptors, fibroblast growth factor receptors (FGFRs), vascular endothelial growth factor receptors, Met (hepatocyte growth factor / scatter factor (HGF / SF) receptor), Ephs (ephrin receptors), and the insulin receptor. RTKs are components of cellular signaling pathways that can be active during embryonic development and adult homeostasis. Because of their roles as growth factor receptors, many RTKs have been implicated in the onset or progression of various cancers, either through receptor gain-of-function mutations or through receptor / ligand overexpression. In some embodiments, the cell surface receptor and / or membrane-associated protein comprises a growth factor receptor. Growth factor receptors can be membrane-bound and can include an extracellular domain, a transmembrane domain, and a cytoplasmic domain. Some non-limiting examples of growth factor receptors are FGFR2B, VEGFR2, PDGFRA, PDGFRB, NGFR, TRKC, TRKB, M6PR, and IGF1R. In some embodiments, the cell surface receptor and / or membrane-associated protein comprises an RTK receptor. In some embodiments, the RTK receptor is an Epidermal Growth Factor Receptor (EGFR). EGFR is an RTK family protein found on the surface of cells. EGFR plays a crucial role in regulating cell growth, survival, and proliferation. In cancer, abnormalities in the EGFR signaling pathway can contribute to uncontrolled cell growth and tumor development. Mutations or overexpression of EGFR can result in continuous activation of the signaling pathway, leading to increased cell division and proliferation. This uncontrolled cell growth can contribute to the formation and progression of cancerous tumors. KRAS is an effector molecule responsible for signal transduction from ligand-bound EGFR to the nucleus. KRAS is an oncogene that regulates cell division and growth. Mutations in the KRAS gene are found in a variety of cancers, including lung, colorectal, and pancreatic cancer, and they are associated with poor prognosis and resistance to certain treatments. For example, activating mutations in KRAS are recognized as a strong predictor of resistance to EGFR-targeted monoclonal antibodies (mAbs).3. Target protein

[0244] Provided herein is a method of degrading a target protein, the method comprising contacting the target protein and a membrane-associated protein (e.g., cell surface receptor) with a binding agent, wherein the contacting of the target protein and the membrane-associated protein with the binding agent leads to degradation of the target protein. In some embodiments, the binding agent(s) disclosed herein comprises a first binding domain that specifically bindsto a target protein. The target protein can be a soluble target protein and a membrane-associated target protein. In some embodiments, the second binding domain of the bispecific binding agents provided herein can bind to an extracellular epitope of a membrane-associated target protein. The binding of the second binding domain to the membrane-associated target protein can result in the internalization of a target cell expressing the membrane-associated target protein.Immune-associated proteins

[0245] In some embodiments, the target protein comprises an immune checkpoint protein, an immunomodulatory protein, an inflammatory cytokine, or an autoantibody.

[0246] In some embodiments, the target protein of the binding agents provided herein can be an immune checkpoint protein. Immune checkpoint proteins are known in the field, and generally refers to proteins that serve as checkpoints produced by some types of immune system cells, such as T cells, and some cancer cells. Some non-limiting examples of immune checkpoint proteins include PD-L1, PD-1, CTLA-4, B7-H3, B7-H4, BTLA, KIR, LAG3, NKG2D, TIM-3, VISTA, SIGLEC6, SIGLEC7, and SIGLEC15. In some cases, the first binding domain can specifically bind to a therapeutic target protein, such as a protein target from a clinically approved antibody (e.g., a target from Table 3).

[0247] In some embodiments, the immune checkpoint protein can be any protein that serves as a cellular checkpoint produced by some type of immune system cell, such as T cells, and some cancer cells. In some embodiments, the immune checkpoint protein comprises PD- Ll, PD-1, CTLA-4, B7-H3, B7-H4, BTLA, KIR, LAG3, NKG2D, TIM-3, VISTA, SIGLEC6, SIGLEC7, or SIGLEC15.

[0248] In some embodiments, the immunomodulatory protein can be any protein that has immunomodulatory activity. In some embodiments, the immunomodulatory protein can have signaling activity upon a certain stimulation that leads to either increased activity of immune cells (i.e., immune activation) or decreased activity of immune cells (i.e., immune suppression). In some embodiments, the immunomodulatory protein can have immune checkpoint activity. In some embodiments, the immunomodulatory protein can overlap with immune checkpoint proteins. In some embodiments, the immunomodulatory protein comprises PD-L1, PD-1, CTLA-4, B7-H3, B7-H4, LAG3, NKG2D, TIM-3, VISTA, CD39, CD73 (NT5E), A2AR, SIGLEC6, SIGLEC7, or SIGLEC15.

[0249] In some embodiments, the target protein comprises an inflammatory cytokine. In some embodiments the inflammatory cytokine comprises interleukin-1 (IL-1), IL-4, IL-10, IL-11, IL-13, IL-12, IL-18, IL-23, IL-6, IL-ip, TGF-P, tumor necrosis factor alpha (TNF-a), interferon gamma (IFNy), or granulocyte-macrophage colony stimulating factor (GM-CSF).

[0250] In some embodiments, the target protein comprises an autoantibody. In some embodiments the autoantibody comprises anti-centromere antibodies, anti-dsDNA antibodies, anti-gp210 antibodies, anti-histone antibodies, anti-Jol antibodies, anti-La / SS-B antibodies, anti-p62 antibodies, anti-PM-Scl antibodies, anti-RNP antibodies, anti-Ro / SS-A antibodies, anti-Sm (Smith) antibodies, anti-splOO antibodies, or anti -topoisomerase antibodies.

[0251] In some embodiments, the target protein can be an inflammation receptor. Some non-limiting exemplary inflammation receptors include TNFR, IL1R, IL2Ralpha, IL2Rbeta. Cell-type specific target proteins

[0252] In some embodiments, the target protein of the binding agents provided herein can be a cancer antigen. In some embodiments, cancer antigens are proteins that are expressed on the surface of certain cancer cells. In other embodiments, cancer antigens are shed by the cancer cells and can be detected in blood and sometimes other body fluids. Thus, cancer antigens can include both cell membrane-associated target proteins and soluble target proteins. Some nonlimiting examples of the cancer antigens include PD-L1, HER2, EGFR, A2AR, CDCP1, MMP14, and TROP2.

[0253] In some embodiments, the target protein can be a B cell antigen. In some instances, the B cell antigen can be a B cell surface marker, e.g., a specific marker of B cell lineage. Some non-limiting examples of B cell antigens include CD 19, CD20, D22, CD23, CD24, CD37, CD40, and HLA-DR. In some embodiments, the target protein can also be a T cell marker. T cell markers can be T cell surface bound or secreted (i.e., extracellular). Some non-limiting examples of T cell markers include CD27, CD28, CD127, PD-1, CD122, CD132, KLRG-1, HLA-DR, CD38, CD69, CDl la, CD58, CD99, CD62L, CD103, CCR4, CCR5, CCR6, CCR9, CCR10, CXCR3, CXCR4, CLA, Granzyme A, Granzyme B, Perforin, CD161, IL-18Ra, c-Kit, and CD130.

[0254] c-Kit, also known as CD117, is a protein that belongs to the receptor tyrosine kinase (RTK) family. It is encoded by the KIT gene and acts as a receptor for a cytokine called stem cell factor (SCF) or mast cell growth factor. The c-Kit receptor plays a role in several cellular processes, including cell survival, proliferation, differentiation, and migration. Upon binding to SCF, c-Kit undergoes dimerization and autophosphorylation, activating various intracellular signaling pathways such as the PI3K-Akt and MAPK-ERK pathways. These signaling pathways are involved in controlling cell growth, survival, and differentiation.

[0255] The c-Kit protein is primarily expressed on the surface of certain cell types, including hematopoietic stem cells, mast cells, melanocytes, and germ cells. In hematopoietic stem cells, c-Kit can play a role in maintaining the sternness and self-renewal capacity of these cells. It also plays a role in the differentiation of hematopoietic cells into various lineages, including red blood cells, white blood cells, and platelets. In mast cells, c-Kit signaling is important for mast cell development, survival, and activation. It regulates mast cell proliferation and differentiation, and its activation leads to the release of histamine, cytokines, and other mediators involved in allergic and immune responses.

[0256] Additionally, c-Kit is involved in the development and migration of melanocytes, which are responsible for producing the pigment melanin. Mutations in the KIT gene can lead to disorders such as piebaldism or familial gastrointestinal stromal tumors (GISTs). In certain embodiments, c-Kit polypeptide and c-Kit protein are used interchangeably to refer to and encompass the polypeptide of UniProt ID: QI 5999. In some embodiments, the target protein is c-Kit (see for example, FIG. 40 - FIG. 48). In some embodiments, the target protein is c- Kit and the membrane-associated protein (e.g., cell surface receptor, internalizing receptor) is CD71 or G7V (see for example, FIG. 40 - FIG. 48).

[0257] CCR5, also known as CC chemokine receptor 5, is a protein that functions as a chemokine receptor on the surface of immune cells. It is encoded by the CCR5 gene and is primarily expressed on the surface of T cells, macrophages, dendritic cells, and other immune cells. CCR5 is important in immune responses and inflammation and can play a role in the migration of immune cells to sites of infection or inflammation. By binding to its ligands, CCR5 promotes the recruitment of immune cells, such as T cells and monocytes, to sites of infection or tissue damage. In certain embodiments, CCR5 polypeptide and CCR5 protein are used interchangeably to refer to and encompass the polypeptide of UniProt ID: B2KKJ9. In some embodiments, the target protein is CCR5. In some embodiments, the target protein is CCR5 and the membrane-associated protein (e.g., cell surface receptor, internalizing receptor) is CD71 (see for example, FIG. 49 - FIG. 52B).

[0258] Other cancer antigens, immuno-modulatory proteins, inflammation receptors, B cell antigens, and T cell marker are known in the field and are also encompassed by the present disclosure. In certain embodiments, some non-limiting examples of the target proteins include PD-L1, HER2, EGFR, PD-1, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, NKG2D, TIM-3, VISTA, LAG3, NKG2D, TIM, SIGLEC6, SIGLEC7, SIGLEC15, CD19, CD20, CDCP1, MMP14, and TROP2.

[0259] In some embodiments, the target protein comprises a tissue-type specific protein. In some embodiments, the target protein comprises a cell-type specific protein. In some embodiments, the target protein comprises an intractable protein and / or a protein that can be pharmacologically difficult to target (e.g., STAT3, TP53, MYC, HLA DQ2.5, GSDMD, TLR7, etc.).Soluble target proteins

[0260] In some embodiments, the target protein of the binding agents provided herein can be a soluble target protein. In some cases, the soluble target proteins include soluble extracellular proteins. For example, the soluble target protein that can be targeted by the binding agents provided herein include an inflammatory cytokine, a growth factor (GF), a toxic enzyme, a target associated with metabolic diseases, a neuronal aggregate, or an autoantibody. These various soluble proteins are known in the art. In some embodiments, non-limiting examples of the inflammatory cytokine include lymphotoxin, interleukin-1 (IL-1), IL-2, IL-5, IL-6, IL- 12, IL-13, IL- 17, IL- 18, IL-23, tumor necrosis factor alpha (TNF-a), interferon gamma (IFNy), and granulocyte-macrophage colony stimulating factor (GM-CSF). In other embodiments, non-limiting examples of the growth factor comprises EGF, FGF, NGF, PDGF, VEGF, IGF, GMCSF, GCSF, TGF, RANK-L, erythropieitn, TPO, BMP, HGF, GDF, neurotrophins, MSF, SGF, GDF, and an isoform thereof. In some embodiments, non-limiting examples of the toxic enzyme comprises a protein arginine deiminase 1 (PAD1), PAD2, PAD3, PAD4, and PAD6, leucocidin, hemolysin, coagulase, treptokinase, hyaluronidase. In certain embodiments, the toxic enzyme comprises PAD2 or PAD4. In some embodiments, the target protein can be associated with a metabolic disease can be PCSK9, HRD1 T2DM, and MOGAT2. In other embodiments, non-limiting examples of the neuronal aggregate comprises A0, TTR, a-synuclein, TAO, and prion. In certain embodiments, the autoantibody comprises IgA, IgE, IgG, IgM, and IgD. Target proteins associated with the conditions described herein are known in the field and new targets are being discovered. In some embodiments, the soluble target protein is selected from one or more of the following: BAFF, IgG4, TN Fa, MICA / B, IL- 23, IL- 1, IL-2, IL-4, IL-6, IL- 12, IFNy, TGF-P, and Ap protein.

[0261] Interleukins are cytokines that play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is to modulate growth, differentiation, and activation during inflammatory and immune responses. In certain non-limiting exemplary embodiments, the interleukin can beIL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL15, IL16, IL17A, IL17B, IL17C, IL17F, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL31, IL32, IL33, IL34, IL36A, IL36B, IL36G, IL36RA, IL37, IL38, ILIA, IL IB, IL1RN. IL-23 (IL23) generally refers to and encompasses a heterodimeric cytokine composed of an IL-12B (IL-12p40) subunit (which is shared with IL-12) and an IL-23A (IL- 23pl9) subunit. In certain instances, IL-23 refers to polypeptides encoded by the IL12B (also known as CLMF, NKSF, CLMF2, IMD28, IMD29, NKSF2, and IL-12B) and the IL23A (also known as P19, SGRF, IL-23, IL-23 A, and IL23P19) genes. In certain instances, IL-23 is part of the IL-12 family of cytokines. In certain instances, defects and / or imbalance in IL-23 is associated with autoimmune diseases and cancer. In certain embodiments, the IL12B gene refers to NCBI Gene ID: 3593 and / or HGNC: 5970. In certain embodiments, IL-12B polypeptide and IL-12B protein are used interchangeably to refer to and encompass the polypeptide of UniProt ID: P29460. In certain embodiments, the IL23A gene refers to NCBI Gene ID: 51561 and / or HGNC: 15488. In certain embodiments, IL-23A polypeptide and IL- 23 A protein are used interchangeably to refer to and encompass the polypeptide of UniProt ID: Q9NPF7. In some embodiments, the target protein is IL-23 and the membrane-associated protein (e.g., cell surface receptor, internalizing receptor) is on cancer cells, specific T cells, or specific innate immune cells (see for example, FIG. 15A). In some embodiments, the target protein is IL-23 and the membrane-associated protein (e.g., cell surface receptor, internalizing receptor) is CD71 (see for example, FIG. 15B).

[0262] In some embodiments, the target protein comprises an immunoglobulin protein. In some embodiments, the immunoglobulin protein is a human immunoglobulin protein. Immunoglobulins can serve various functions in the immune system. For example, immunoglobulins can recognize and bind specifically to antigens, prevent viruses from entering and infecting host cells, block the binding of toxins to their target cells, or directly attack and destroy bacteria. Immunoglobulins can also activate other cells of the immune system, such as macrophages, neutrophils, and natural killer cells, and can modulate the immune response by interacting with various immune cells and signaling pathways. In some embodiments, the target protein can be IgG, IgA, IgM, IgD, or IgE. In some embodiments, the target protein can be IgG. In some embodiments, the target protein can be IgGl, IgG2, IgG3, or IgG4. In some embodiments, the target protein is IgG4. In certain embodiments, the IgG4 polypeptide and the IgG4 protein are used interchangeably to refer to and encompass the polypeptide of UniProt ID: P01861. In some embodiments, the target protein is IgG4 and the membrane-associated protein (e.g., cell surface receptor, internalizing receptor) is CD71 (seefor example, FIG. 15B, and FIG. 17A - FIG. 20B). In some embodiments, the target protein is IgG4 and the membrane-associated protein (e.g., cell surface receptor, internalizing receptor) is EphA2 or CD71 (see for example, FIG. 15C).

[0263] In some embodiments, the target protein comprises a neuronal aggregate protein. In some embodiments the neuronal aggregate protein comprises amorphous aggregates, oligomers, amyloid fibrils, or A0 protein (see Example 8). A0 protein (also known as Amyloid P or Abeta) generally refers to and encompasses peptides of about 36 to about 43 amino acids that are the main component of amyloid plaques. In certain embodiments, AJ3 protein refers to an AJ3 polypeptide, an A|3 peptide and / or an Ap molecule. In certain instances, Ap peptides are derived from the amyloid-P precursor protein (also known as APP, AAA, ADI, PN2, ABPP, APPI, CVAP, ABETA, PN-II, preA4, CTFgamma, and alpha-sAPP). In certain instances, Ap peptides can aggregate to form flexible soluble oligomers which may exist in several forms. In certain instances, Ap can form deposits, amyloid plaques, and / or take a misfolded oligomeric form. In certain embodiments, Ap protein is associated with Alzheimer's disease and / or cerebral amyloid angiopathy. In certain embodiments, the APP gene refers to NCBI Gene ID: 351 and / or HGNC: 620. In certain embodiments, the amyloid-beta precursor polypeptide and the amyloid-beta precursor protein are used interchangeably to refer to and encompass the polypeptide of UniProt ID: P05067. In some embodiments, the target protein is Ap aggregated protein (e.g., amyloid beta fibrils) and the membrane-associated protein is an internalizing receptor (see for example, FIG. 27A). In some embodiments, the target protein is Ap aggregated protein (e.g., amyloid beta fibrils) and the membrane-associated protein (e.g., cell surface receptor, internalizing receptor) is CD71 (see for example, FIG. 28 - FIG. 29F). In some embodiments, the target protein is Ap aggregated protein (e.g., amyloid beta fibrils) and the membrane-associated protein (e.g., cell surface receptor, internalizing receptor) is CD163 (see for example, FIG. 30A - FIG. 31).

[0264] Interferons belong to the large class of proteins known as cytokines and are made and released by host cells in response to the presence of several viruses. More than twenty distinct IFN genes and proteins have been identified in animals, including humans. They are typically divided among three classes: Type I IFN, Type II IFN, and Type III IFN. In certain non-limiting exemplary embodiments, the interferon can be IFNA, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA14, IFNA16, IFNA17, IFNA21, IFNB, and IFNG.

[0265] Tumor necrosis factors are multifunctional cytokines that play important roles in diverse cellular events such as cell survival, proliferation, differentiation, and death. In certain non-limiting exemplary embodiments, the tumor necrosis factors can be TNFA, TNFB, TRAIL, TL1, BAFF, APRIL, RANKL, CD40LG, EDA, FASLG, CD70.

[0266] Transforming growth factor betas are multifunctional cytokine belonging to the transforming growth factor superfamily that includes three different mammalian isoforms (TGFB1, TGFB2, TGFB3) and many other signaling proteins. Among its key functions is regulation of inflammatory processes and playing a role in stem cell differentiation as well as T-cell regulation and differentiation. In certain non-limiting exemplary embodiments, the TGF- beta can be TGFB1, TGFB2, TGFB3, GDF15, GDF2, BMP10, INHA, and BMP3.Membrane-bound and / or cell-surface target proteins

[0267] In some embodiments, the target protein comprises a membrane-bound protein. In some embodiments, the membrane-bound target protein comprises multiple ligand-binding sites. In some embodiments, the membrane-bound target protein comprises unknown ligandbinding sites. In some embodiments, the membrane-bound target protein is selected from one or more of the following: MerTK, PD-1, ICOS, IgG4, TIM3, B7H4, RAGE, HLA DQ2.5, GSDMD, TLR7, CD86, MHC-II, MHC-I, ICAM, CD83, CD3i and a4 integrin protein.

[0268] In some embodiments, the membrane-bound target protein is ICOS (see Example4). ICOS (Inducible T-cell COStimulator) generally refers to and encompasses a CD28- superfamily costimulatory molecule that is expressed on activated T cells. In certain instances, ICOS refers to a polypeptide encoded by the ICOS (also known as AILIM, CD278, and CVID1) gene. In certain instances, ICOS is part of the CD28 and CTLA-4 cell-surface receptor family. In certain instances, defects and / or imbalance in ICOS is associated with primary immunodeficiency characterized by antibody deficiency, hypogammaglobulinemia, recurrent bacterial infections and an inability to mount an antibody response to antigen. In certain embodiments, the ICOS gene refers to NCBI Gene ID: 29851 and / or HGNC: 5351. In certain embodiments, ICOS polypeptide and ICOS protein are used interchangeably to refer to and encompass the polypeptide of UniProt ID: Q9Y6W8.

[0269] In some embodiments, the membrane-bound target protein is PD-1 (see Example5). PD-1 (Programmed cell death protein 1) generally refers to and encompasses a cell surface receptor encoded, in humans, by the PDCD1 (also known as PD1, PD-1, CD279, SLEB2, hPD- 1, hPD-1, and hSLEl) gene. In certain instances, PD-1 is part of the immunoglobulin superfamily. In certain instances, PD-1 is expressed on T cells and pro-B cells. In certain instances, PD-1 is highly expressed in several cancers, and is associated with cancer immuneevasion. In certain embodiments, the PD-1 gene refers to NCBI Gene ID: 5133 and / or HGNC: 8760. In certain embodiments, PD-1 polypeptide and PD-1 protein are used interchangeably to refer to and encompass the polypeptide of UniProt ID: Q15116. In some embodiments, the target protein is PD-1. In some embodiments, the target protein is PD-1 and the membrane- associated protein (e.g., cell surface receptor, internalizing receptor) is CD30 (see for example, FIG. 6A - FIG. 7B) In some embodiments, the target protein is PD-1 and the membrane- associated protein (e.g., cell surface receptor, internalizing receptor) is 0X40 (see for example, FIG. 8A and FIG. 8B). In some embodiments, the target protein is PD-1 and the membrane- associated protein (e.g., cell surface receptor, internalizing receptor) is CCR4 (see for example, FIG. 10A) In some embodiments, the target protein is PD-1 and the membrane-associated protein (e.g., cell surface receptor, internalizing receptor) is CCR8 (see for example, FIG. 10B) In some embodiments, the target protein is PD-1 and the membrane-associated protein (e.g., cell surface receptor, internalizing receptor) is WT IL-2 or IL-2 mutein (see for example, FIG. 13A and FIG. 13B)

[0270] In some embodiments, the membrane-bound target protein is RAGE (see Example 6). RAGE (receptor for advanced glycation end products) generally refers to and encompasses a cell surface receptor encoded in humans by the AGER (also known as RAGE, sRAGE; SCARJ1) gene. In certain instances, RAGE is part of the immunoglobulin superfamily. In certain instances, RAGE is a transmembrane receptor. In certain instances, high expression of RAGE is associated with inflammatory conditions. In certain instances, defects and / or imbalance in RAGE is associated with Alzheimer's disease, arthritis, atherosclerosis, congestive heart failure, diabetes, myocardial infarction, psoriasis, rheumatoid arthritis, and Takayasu's arteritis. In certain embodiments, the AGER gene refers to NCBI Gene ID: 177 and / or HGNC: 320. In certain embodiments, RAGE polypeptide and RAGE protein are used interchangeably to refer to and encompass the polypeptide of UniProt ID: Q15109. In some embodiments, the target protein is RAGE and the membrane-associated protein (e.g., cell surface receptor, internalizing receptor) is specific to inflamed tissues (see for example, FIG.14)Intracellular target proteins

[0271] In some embodiments, the target protein comprises an intracellular protein. In some embodiments, the intracellular protein is a membrane-associated (e.g., membrane-bound) intracellular protein. For example, the intracellular protein can be an endosomal-associated protein, such as TLR7 (see Example 9). Endosomes can refer to membrane-bound compartments within cells responsible for processing and degrading various cellularcomponents. The endosomal degradation pathway can be leveraged to regulate TLR7 levels and activity. For example, the binding agent(s) disclosed herein can specifically target endosomal-associated proteins, leading to the degradation of the protein within the endosome.

[0272] Several proteins associated with endosomes (e.g., TLR7, PSEN1, PSEN2, LRRK2, TPC2, APPL1, VPS35, ESCRT, etc.) have been implicated in various diseases. For example, mutations in presenilin 1 and 2 (PSEN1, PSEN2) have been associated with early-onset familial Alzheimer's disease (FAD). Presenilins are involved in the processing of amyloid precursor protein (APP) and play a role in the generation of amyloid-beta peptides, which are central to Alzheimer's pathology. Mutations in LRRK2 gene are associated with Parkinson's disease. LRRK2 is implicated in regulating vesicular trafficking, and its dysfunction may impact endosomal functions and contribute to neurodegeneration. Mutations in TPC2 (Two-Pore Channel 2) have been associated with the development of obesity and metabolic syndrome. TPC2 regulates endolysosomal calcium release and is involved in the regulation of cellular metabolism. Adaptor protein, phosphotyrosine interacting with PH domain, and leucine zipper 1 (APPL1) is linked to insulin resistance and type 2 diabetes. APPL1 is involved in endosomal trafficking of insulin receptors and insulin signaling. Mutations in VPS35 are associated with autosomal dominant Parkinson's disease. VPS35 is a component of the retromer complex involved in endosomal sorting and retrieval of membrane proteins. Dysfunction of various ESCRT complex proteins, such as CHMP2B, has been implicated in neurodegenerative diseases like frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In some embodiments, the intracellular target protein is selected from one or more of the following: TLR7, Tau, PSEN1, PSEN2, LRRK2, TPC2, APPL1, VPS35, and ESCRT. In some embodiments, the target protein is TLR7. In some embodiments, the target protein is TLR7 and the membrane-associated protein (e.g., cell surface receptor, internalizing receptor) is CD71 (see for example, FIG. 32A - FIG. 36C).Modulating Intracellular Downstream Cell Signaling Proteins

[0273] In some cases, the objective is to modulate an intracellular protein through targeting upstream cell surface receptors. In some embodiments, the intracellular protein is a downstream cell signaling protein or a receptor-coupled protein. In some embodiments, the target protein comprises a surface protein or a downstream cell signaling protein. In some embodiments, the cell surface protein or the downstream cell signaling protein is selected from the group consisting of EGFR receptors, HER receptors, PDGF receptors, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lek, Fgr, Yes, Csk,Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, TNFp receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3 / 6, MKK4 / 7, ASKl,Cot, NIK, Bub, Myt 1, Weel, Casein kinases, PDK1, SGK1, SGK2, SGK3, Aktl, Akt2, Akt3, p90Rsks, p70S6 Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs, Chkl, Chk2, LKB-1, MAPKAPKs, Piml, Pim2, Pim3, IKKs, Cdks, Inks, Erks, IKKs, GSK3a, GSK3P, Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class 3, mTor, SAPK / JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR, Receptor protein tyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs), Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, Low molecular weight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C, PPI, PP5, inositol phosphatases, PTEN, SHIPs, myotubularins, phosphoinositide kinases, phopsholipases, prostaglandin synthases, 5 -lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor / scaffold proteins, She, Grb2, BLNK, LAT, B cell adaptor for PI3- kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nek, Grb2 associated binder (GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-Cell leukemia family, IL-2, IL-4, IL-8, IL-6, interferon y, interferon a, suppressors of cytokine signaling (SOCs), Cbl, SCF ubiquitination ligase complex, APC / C, adhesion molecules, integrins, Immunoglobulin- like adhesion molecules, selectins, cadherins, catenins, focal adhesion kinase, pl30CAS, fodrin, actin, paxillin, myosin, myosin binding proteins, tubulin, eg5 / KSP, CENPs, P- adrenergic receptors, muscarinic receptors, adenylyl cyclase receptors, small molecular weight GTPases, H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam, Sos, Dbl, PRK, TSC1,2, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Bcl-2, Mcl-1, Bcl-XL, Bcl-w, Bcl-B, Al, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma, IAPS, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdkl, Cdk 7, Cyclin D, Cyclin E, Cyclin A, Cyclin B, Rb, pl 6, pl4Arf, p27KIP, p21CIP, molecular chaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoA Carboxylase, ATP citrate lyase, nitric oxide synthase, caveolins, endosomal sorting complex required for transport (ESCRT) proteins, vesicular protein sorting (Vsps), hydroxylases, prolyl-hydroxylases PHD- 1, 2 and 3, asparagine hydroxylase FH4 transferases, Pinl prolyl isomerase, topoisomerases, deacetylases, Histone deacetylases, sirtuins, histone acetylases, CBP / P300 family, MYST family, ATF2, DNA methyl transferases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, VHL, WT-1, p53, Hdm, PTEN, ubiquitin proteases, urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) system, cathepsins, metalloproteinases, esterases,hydrolases, separase, potassium channels, sodium channels, , multi-drug resistance proteins, P- Gycoprotein, nucleoside transporters, Ets, Elk, SMADs, Rel-A (p65-NFKB), CREB, NF AT, ATF-2, AFT, Myc, Fos, Spl, Egr-1, T-bet, P-catenin, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr- 1, P-catenin, FOXO STAT1, STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-1, HMGA, pS6, 4EPB-1, eIF4E-binding protein, RNA polymerase, initiation factors, elongation factors.

[0274] In some embodiments, the target protein comprises a mutation. In some embodiments, the target protein comprising a mutation is a surface protein or a downstream cell signaling protein. For example, the cell surface protein comprises a mutation or the downstream cell signaling protein comprises a mutation (see Example 10, and FIG. 37 - FIG. 39B). In some embodiments, the mutation can be an oncogenic mutation. Oncogenic mutations can refer to specific genetic alterations in the DNA of a cell that can lead to the development of cancer. There are different types of oncogenic mutations, including but not limited to, point mutations, gene amplification, gene fusion, and chromosomal rearrangements. These mutations can occur in various genes involved in regulating cell growth, division, and death. When these genes are altered, they can disrupt normal cellular processes, leading to uncontrolled cell growth and the formation of a tumor. For example, mutations that lead to EGFR overexpression (known as upregulation or amplification) have been associated with a number of cancers, including adenocarcinoma of the lung cancer, anal cancers, glioblastoma and epithelian tumors of the head and neck. Mutations, amplifications or misregulations of EGFR or family members are implicated in about 30% of all epithelial cancers. Many of these somatic mutations involving EGFR led to its constant activation, which produces uncontrolled cell division. Therefore, the degradation of EGFR in cancer is a promising treatment modality for cancer. In some embodiments, the cell surface protein is an EGFR protein. In some embodiments, the EGFR protein comprises a mutation, such as an oncogenic mutation. In some embodiments, the modulation of upstream cell surface proteins affect the scaffolding for downstream cell signaling proteins such as a Ras protein. In some embodiments, the Ras protein comprises a mutation, such as an oncogenic mutation. In some cases, the binding agent(s) disclosed herein can drive inhibition of signaling pathways, such as ERK signaling which is downstream of K-Ras. In some cases, the binding agent(s) disclosed herein can drive inhibition of signaling pathways independent of EGFR mutation status. In some cases, the binding agent(s) disclosed herein can drive inhibition of signaling pathways independent of K- Ras mutation status. In some cases, the binding agent(s) disclosed herein can drive tumor cell killing independent of K-Ras mutation status.

[0275] Signaling pathways and their members have been described (see, for example, Hunter T. Cell Jan. 7, 2000; 100(1): 13-27, which is herein incorporated by reference in its entirety). Exemplary signaling pathways include the following pathways and their members: The MAP kinase pathway including Ras, Raf, MEK, ERK and elk; the PI3K / Akt pathway including PI-3-kinase (PI3K), PDK1, Akt and Bad; the NF-KB pathway including IKKs, IkB; the Wnt pathway including frizzled receptors, beta-catenin, APC and other co-factors and TCF; the T cell receptor (TCR) pathway including Lek and Zap70; and the B cell receptor (BCR) pathway including Lyn, Syk, and PLCy2. In some embodiments of the invention, the signaling proteins to be modulated are members of the MAP kinase, Akt, NFkB, WNT, RAS / RAF / MEK / ERK, JNK / SAPK, p38 MAPK, Src Family Kinases, JAK / STAT, TCR, BCR, and / or PKC signaling pathways.

[0276] In some embodiments, Ras protein is selected from K-Ras, N-Ras, or H-Ras. Ras proteins are small guanine nucleotide-binding proteins that act as molecular switches by cycling between active GTP-bound and inactive GDP -bound conformations. Ras signaling is regulated through a balance between activation by guanine nucleotide exchange factors (GEFs), most commonly son of sevenless (SOS), and inactivation by GTPase-activating proteins (GAPs). The Ras proteins play a critical role in the regulation of cell proliferation, differentiation, and survival. Dysregulation of the Ras signaling pathway is almost invariably associated with disease. Hyper-activating somatic mutations in Ras are among the most common lesions found in human cancer. Most of these mutations have been shown to decrease the sensitivity of Ras to GAP stimulation and decrease its intrinsic GTPase activity, leading to an increase in the active GTP-bound population. Although mutation of any one of the three Ras isoforms (K-Ras, N-Ras, or H-Ras) has been shown to lead to oncogenic transformation, K- Ras mutations are by far the most common in human cancer. For example, K-Ras mutations are known to be often associated with pancreatic, colorectal and non-small-cell lung carcinomas. Similarly, H-Ras mutations are common in cancers such as papillary thyroid cancer, lung cancers and skin cancers, while N-Ras mutations occur frequently in hepatocellular carcinoma.4. Target cell

[0277] Provided herein is a method of degrading a target protein, the method comprising contacting the target protein and a membrane-associated protein with a binding agent, wherein the membrane-associated protein is associated with a target cell.

[0278] In some embodiments, the target cell comprises any cell expressing a membrane- associated protein. In some embodiments, the target cell can be an immune cell. In some embodiments, the immune cell can be a monocyte, a macrophage, a lymphocyte (e.g., natural killer cells, T cells, and B cells), and / or a monocytes. In some embodiments, the target cell comprises a microglia cell, endothelial cell, immune cell, innate immune cell, regulatory T cell, or T cell.

[0279] In some embodiments, the target cell comprises a microglia cell, endothelial cell, smooth muscle cell, immune cell, plasma cell, B cell, neuronal cell, innate immune cell, lymphocyte, monocyte, granulocyte, regulatory T cell, T cell, effector T cell, activated CD8 T cell, CD8+ T cell, CD4+ T cell, Th 17 cell, macrophage, or dendritic cell.

[0280] Without being bound by theory, in the cases where the target protein is a membrane- associated target protein, the target cell needs to express both the membrane-associated target protein and the endogenous cell surface receptor. For instance, a binding agent provided herein can comprise (1) a first binding domain which has CXCL12 Fc or a variant thereof that specifically binds to CXCR4 and / or CXCR7, and (2) a second binding domain which includes a Fab targeting PD-L1. In this case, the target cell needs to express (1) CXCR4 and / or CXCR7 and (2) PD-L1.

[0281] As mentioned above, in some embodiments, the bispecific binding agents of the present disclosure can specifically bind to an extracellular epitope of a membrane-associated target protein, and such binding can result in the membrane-associated target protein bound to the bispecific binding agent. Thus, one skilled in the art would appreciate that any cell expressing a target protein could be a target cell for the purpose of the present disclosure. For example, the target cells encompassed by the present can be a neoplastic cell. A neoplasm is an abnormal growth of cells. Neoplastic cells are cells that are undergoing or have undergone an abnormal growth. In some instances, these abnormally growing cells can cause tumor growth and can be both benign and malignant.

[0282] Alternatively, the target cells encompassed by the present disclosure can be cancer cells. Some non-limiting examples of target cells include cancer cells, such as cells from breast cancer, B cell lymphoma, pancreatic cancer, Hodgkin’s lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin’s B-cell (B-NHL), melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, and colorectal cancer.

[0283] In other embodiments, the target cells can be immune cells. For instance, the immune cells can be monocytes, macrophages, lymphocytes (e.g., natural killer cells, T cells, and B cells), and monocytes.II.METHODS OF MAKING

[0284] Provided herein are methods of making the bispecific binding agent disclosed herein. Exemplary structures of binding agents defined herein are described throughout. Exemplary structures are further described in: Weidle U et al. (2013) The Intriguing Options of Multispecific Antibody Formats for Treatment of Cancer. Cancer Genomics & Proteomics 10: 1-18 (2013); and Spiess C et al. (2015) Alternative molecular formats and therapeutic applications for bispecific antibodies. Molecular Immunology 67: 95-106; the full contents of each of which is incorporated by reference herein).

[0285] In some embodiments, binding agents disclosed herein can comprise more than one antigen-binding site, where different sites are specific for different antigens. In some embodiments, multifunctional or multispecific antibody binding agents can bind more than one (e.g., two or more) epitopes on the same antigen. In some embodiments, multifunctional or multispecific antibody binding agents comprise an antigen-binding site specific for a target cell (e.g., cancer cell) and a different antigen-binding site specific for an immune effector cell. In some embodiments, the multifunctional or multispecific antibody binding agent is a bispecific antibody binding agent. Bispecific antibody binding agents can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG appended with an additional antigen-binding moiety; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates.

[0286] BsIgG is a format that is monovalent for each antigen. Exemplary BsIgG formats include but are not limited to crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT- IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, kl-body, orthogonal Fab. See Spiess et al. Mol. Immunol. 67(2015):95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which contains an anti-CD3 arm and an anti-EpCAM arm; and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2. In some embodiments, BsIgG comprises heavy chains that are engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using a “knobs-into-holes” strategy, a SEED platform, a common heavy chain (e.g., in kl-bodies), and use of heterodimeric Fc regions. See Spiess et al. Mol. Immunol. 67(2015):95-106. Strategies that have been used to avoid heavychain pairing of homodimers in BsIgG include knobs-in-holes, duobody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity. BsIgG can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly into a BsIgG. BsIgG can also be produced by expression of the component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, e.g., using protein A and sequential pH elution.

[0287] IgG appended with an additional antigen-binding moiety is another format of bispecific antibody binding agents. For example, monovalent IgG can be engineered to have bispecificity by appending an additional antigen-binding unit onto the monovalent IgG, e.g., at the N- or C- terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy chain or variable light chain), engineered protein scaffolds, and paired antibody variable domains (e.g., single chain variable fragments or variable fragments). Examples of appended IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol. Immunol. 67(2015):95-106. An example of an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (Abb Vie), which binds IL-la and IL-1P; and ABT-122 (Abb Vie), which binds TNF and IL-17A.

[0288] Bispecific antibody fragments (BsAb) are a format of bispecific antibody binding agents that lack some or all of the antibody constant domains. For example, some BsAb lack an Fc region. In some embodiments, bispecific antibody fragments include heavy and light chain regions that are connected by a peptide linker that permits efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include but are not limited to nanobody, nanobody-HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab- scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intrabody. For example, the BiTE format comprises tandem scFvs, where the component scFvs bind to a surface antigen on cancer cells.

[0289] Bispecific fusion proteins include antibody fragments linked to other proteins, e.g., to add additional specificity and / or functionality. An example of a bispecific fusion protein is an immTAC, which comprises an anti-CD3 scFv linked to an affinity-matured T-cell receptor that recognizes HLA-presented peptides. In some embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody binding agents with higher valency. Also,fusions to albumin binding proteins or human serum albumin can be extend the serum half-life of antibody fragments.

[0290] In some embodiments, chemical conjugation, e.g., chemical conjugation of antibodies and / or antibody fragments, can be used to create BsAb molecules. An exemplary bispecific antibody conjugate includes the CovX-body format, in which a low molecular weight drug is conjugated site-specifically to a single reactive lysine in each Fab arm or an antibody or fragment thereof. In some embodiments, the conjugation improves the serum half-life of the low molecular weight drug. An exemplary CovX-body is CVX-241 (NCTO 1004822), which comprises an antibody conjugated to two short peptides inhibiting either VEGF or Ang2.

[0291] The antibody binding agents can be produced by recombinant expression, e.g., of at least one or more component, in a host system. Exemplary host systems include eukaryotic cells (e.g., mammalian cells, e.g., CHO cells, or insect cells, e.g., SF9 or S2 cells) and prokaryotic cells (e.g., E. coli). Bispecific antibody binding agents can be produced by separate expression of the components in different host cells and subsequent purification / assembly. Alternatively, the antibody binding agents can be produced by expression of the components in a single host cell. Purification of bispecific antibody binding agents can be performed by various methods such as affinity chromatography, e.g., using protein A and sequential pH elution. In other embodiments, affinity tags can be used for purification, e.g., histidine- containing tag, myc tag, or streptavidin tag.

[0292] Various methods of producing multispecific antibodies have been disclosed to address the problem of incorrect heavy chain pairing. Exemplary methods are described below. Exemplary multispecific antibody formats and methods of making said multispecific antibodies are also disclosed in e.g., Speiss et al. Molecular Immunology 67 (2015) 95-106; and Klein et al mAbs 4:6, 653-663; November / December 2012; the entire contents of each of which are incorporated by reference herein.

[0293] Heterodimerized bispecific antibodies are based on the natural IgG structure, wherein the two binding arms recognize different antigens. IgG derived formats that enable defined monovalent (and simultaneous) antigen binding are generated by forced heavy chain heterodimerization, combined with technologies that minimize light chain mispairing (e.g., common light chain). Forced heavy chain heterodimerization can be obtained using, e.g., knobin-hole OR strand exchange engineered domains (SEED).Knob-in-Hole

[0294] Knob-in-Hole as described in US 5,731,116, US 7,476,724 and Ridgway, J. et al. (1996) Prot. Engineering 9(7): 617-621, broadly involves: (1) mutating the CH3 domain of oneor both antibodies to promote heterodimerization; and (2) combining the mutated antibodies under conditions that promote heterodimerization. “Knobs” or “protuberances” are typically created by replacing a small amino acid in a parental antibody with a larger amino acid (e.g., T366Y or T366W); “Holes” or “cavities” are created by replacing a larger residue in a parental antibody with a smaller amino acid (e.g., Y407T, T366S, L368A and / or Y407V).

[0295] For bispecific antibodies including an Fc domain, introduction of specific mutations into the constant region of the heavy chains to promote the correct heterodimerization of the Fc portion can be utilized. Several such techniques are reviewed in Klein et al. (mAbs (2012) 4:6, 1-11), the contents of which are incorporated herein by reference in their entirety. These techniques include the “knobs-into-holes” (KiH) approach which involves the introduction of a bulky residue into one of the CH3 domains of one of the antibody heavy chains. This bulky residue fits into a complementary “hole” in the other CH3 domain of the paired heavy chain so as to promote correct pairing of heavy chains (see e.g., US7642228).

[0296] Exemplary Fc mutations are provided by Igawa and Tsunoda who identified 3 negatively charged residues in the CH3 domain of one chain that pair with three positively charged residues in the CH3 domain of the other chain. These specific charged residue pairs are: E356-K439, E357-K370, D399-K409 and vice versa. By introducing at least two of the following three mutations in chain A: E356K, E357K and D399K, as well as K370E, K409D,K439E in chain B, alone or in combination with newly identified disulfide bridges, they were able to favor very efficient heterodimerization while suppressing homodimerization at the same time (Martens T et al. A novel one-armed antic- Met antibody inhibits glioblastoma growth in vivo. Clin Cancer Res 2006; 12:6144-52; PMID: 17062691). Xencor defined 41 variant pairs based on combining structural calculations and sequence information that were subsequently screened for maximal heterodimerization, defining the combination of S364H, F405A (HA) on chain A and Y349T, T394F on chain B (TF) (Moore GL et al. A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. MAbs 2011; 3:546-57; PMID: 22123055).

[0297] Other exemplary Fc mutations to promote heterodimerization of multispecific antibodies include those described in the following references, the contents of each of which is incorporated by reference herein, WO2016071377A1, US20140079689A1,US20160194389A1, US20160257763, WO2016071376A2, W02015107026A1,W02015107025A1, W02015107015A1, US20150353636A1, US20140199294A1,US7750128B2, US20160229915 Al, US20150344570A1, US8003774A1,US20150337049A1, US20150175707A1, US20140242075A1, US20130195849A1,US20120149876A1, US20140200331A1, US9309311B2, US8586713, US20140037621A1, US20130178605A1, US20140363426A1, US20140051835A1 and US20110054151A1.

[0298] Stabilizing cysteine mutations have also been used in combination with KiH and other Fc heterodimerization promoting variants, see e.g., US7183076. Other exemplary cysteine modifications include, e.g., those disclosed in US20140348839A1, US7855275B2, and US9000130B2.Strand Exchange Engineered Domains (SEED)

[0299] Heterodimeric Fc platform that support the design of bispecific and asymmetric fusion proteins by devising strand-exchange engineered domain (SEED) C(H)3 heterodimers are known. These derivatives of human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers that are composed of alternating segments of human IgA and IgG C(H)3 sequences. The resulting pair of SEED C(H)3 domains preferentially associates to form heterodimers when expressed in mammalian cells. SEEDbody (Sb) fusion proteins consist of [IgGl hinge]-C(H)2-[SEED C(H)3], that may be genetically linked to one or more fusion partners (see e.g., Davis JH et al. SEEDbodies: fusion proteins based on strand exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. Protein Eng Des Sei 2010; 23: 195-202; PMID:20299542 and US8871912. The contents of each of which are incorporated by reference herein).Fc-containing entities (mini-antibodies)

[0300] Fc-containing entities, also known as mini-antibodies, can be generated by fusing scFv to the C-termini of constant heavy region domain 3 (CH3-scFv) and / or to the hinge region (scFv-hinge-Fc) of an antibody with a different specificity. Trivalent entities can also be made which have disulfide stabilized variable domains (without peptide linker) fused to the C- terminus of CH3 domains of IgGs.Duobody

[0301] “Duobody” technology to produce bispecific antibodies with correct heavy chain pairing are known. The DuoBody technology involves three basic steps to generate stable bispecific human IgGl antibodies in a post-production exchange reaction. In a first step, two IgGls, each containing single matched mutations in the third constant (CH3) domain, are produced separately using standard mammalian recombinant cell lines. Subsequently, these IgGl antibodies are purified according to standard processes for recovery and purification. After production and purification (post-production), the two antibodies are recombined under tailored laboratory conditions resulting in a bispecific antibody product with a very high yield(typically >95%) (see e.g., Labrijn et al, PNAS 2013; 110(13):5145-5150 and Labrijn et al. Nature Protocols 2014;9(10):2450-63, the contents of each of which are incorporated by reference herein).Electrostatic Interactions

[0302] Methods of making multifunctional or multispecific antibodies using CH3 amino acid changes with charged amino acids such that homodimer formation is electrostatically unfavorable are disclosed. EP 1870459 and WO 2009089004 describe other strategies for favoring heterodimer formation upon co-expression of different antibody domains in a host cell. In these methods, one or more residues that make up the heavy chain constant domain 3 (CH3), CH3-CH3 interfaces in both CH3 domains are replaced with a charged amino acid such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. Additional methods of making multifunctional or multispecific binding agents using electrostatic interactions are described in the following references, the contents of each of which is incorporated by reference herein, include US20100015133, US8592562B2, US9200060B2, US20140154254A1, and US9358286A1.Common Light Chain

[0303] Light chain mispairing needs to be avoided to generate homogenous preparations of bispecific IgGs. One way to achieve this is through the use of the common light chain principle, i.e. combining two binders that share one light chain but still have separate specificities. An exemplary method of enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a common variable light chain to interact with each of the heteromeric variable heavy chain regions of the bispecific antibody. Compositions and methods of producing bispecific antibodies with a common light chain as disclosed in, e.g., US7183076B2, US20110177073 Al, EP2847231A1, W02016079081A1, and EP3055329A1, the contents of each of which is incorporated by reference herein.CrossMab

[0304] Another option to reduce light chain mispairing is the CrossMab technology which avoids non-specific L chain mispairing by exchanging CHI and CL domains in the Fab of one half of the bispecific antibody. Such crossover variants retain binding specificity and affinity, but make the two arms so different that L chain mispairing is prevented. The CrossMab technology (as reviewed in Klein et al. Supra) involves domain swapping between heavy and light chains so as to promote the formation of the correct pairings. Briefly, to construct a bispecific IgG-like CrossMab antibody that could bind to two antigens by using two distinct light chain-heavy chain pairs, a two-step modification process is applied. First, a dimerizationinterface is engineered into the C-terminus of each heavy chain using a heterodimerization approach, e.g., Knob-into-hole (KiH) technology, to ensure that only a heterodimer of two distinct heavy chains from one antibody (e.g., Antibody A) and a second antibody (e.g., Antibody B) is efficiently formed. Next, the constant heavy 1 (CHI) and constant light (CL) domains of one antibody are exchanged (Antibody A), keeping the variable heavy (VH) and variable light (VL) domains consistent. The exchange of the CHI and CL domains ensured that the modified antibody (Antibody A) light chain would only efficiently dimerize with the modified antibody (antibody A) heavy chain, while the unmodified antibody (Antibody B) light chain would only efficiently dimerize with the unmodified antibody (Antibody B) heavy chain; and thus only the desired bispecific CrossMab would be efficiently formed (see e.g., Cain, C. SciBX 4(28); doi : 10.1038 / scibx.2011.783, the contents of which are incorporated by reference herein).Common Heavy Chain

[0305] An exemplary method of enhancing the formation of a desired bi specific antibody from a mixture of monomers is by providing a common variable heavy chain to interact with each of the heteromeric variable light chain regions of the bispecific antibody. Compositions and methods of producing bispecific antibodies with a common heavy chain are disclosed in, e.g., US20120184716, US20130317200, and US20160264685 Al, the contents of each of which is incorporated by reference herein. Expression of two different heavy and light chains in a single cell can create misassembled unwanted species, such as heavy-light chain mispairing. These impurities can be difficult to remove due to their similarity to the correct format. Enhancing the formation of a desired bispecific antibody from a mixture of monomers can also be achieved through correct heavy -light chain pairing. For example, bYlok® bispecific pairing technology can be used to engineer differential cysteine binding between the light and heavy chain to help with correct pairing (e.g., engineering a native disulfide bridge and relocating it from one of the constant domains, such as CH1 / CL, to the variable domains, such as VH / VL). In some cases, one or more mutations can be incorporated into the binding agents disclosed herein. In some embodiments, the one or more mutations are configured to improve scFv stability and / or create diabodies. In some embodiments, the one or more mutations are configured to drive correct heavy -light chain pairing. In some embodiments, a native disulfide bridge between a CHI region and a CL region is relocated to be between a VH region and a VL region. In some embodiments, a native CHI region cysteine and a native CL region cysteine are relocated to a VH region and a VL region, respectively.

[0306] In some embodiments, the binding agent comprises: (a) a first CHI domain (CHI) and a first CL domain (CL), the first CHI domain and the first CL domain interacting together at a first CHCL interface to form a first CHCL domain (CHCL); (b) a second CHI domain (CHI) and a second CL domain (CL), the second CHI domain and the second CL domain interacting together at a second CHCL interface to form a second CHCL domain (CHCL). In some embodiments, the first CHI domain and / or the second CHI domain have at least one mutation relative to a human immunoglobulin CHI domain, referred to as the CHI mutant residue(s). In some embodiments, each CHI mutant residue is only present in one of first CHI domain or the second CHI domain. In some embodiments, the first CL domain and / or the second CL domain have at least one mutation relative to a human immunoglobulin CL domain, referred to as the CL mutant residue(s). In some embodiments, each CL mutant residue is only present in one of first CL domain or the second CL domain. In some embodiments, the CHI mutant residue(s) and the CL mutant residue(s) comprise charged amino acids such that a first CHI mutant residue and a first CL mutant residue comprise a charge pair. In some embodiments, the CHI mutant residues and the CL mutant residues comprise a steric pair such that (a) (i) the second CHI mutant residue has steric conflict with the first CL domain or the second CL domain or (ii) the second CL mutant residue has steric conflict with the first CHI domain or the second CHI domain and (b) the second CHI mutant residue and the second CL mutant residue do not have steric conflict. In some embodiments, the first CHI domain is attached to a first variable heavy domain (VH), and the first CL domain is attached to a first variable light domain (VL), and the second CHI domain is attached to a second VH domain, and the second CL domain is attached to a second VL domain, such that when combined, the first VH domain, first VL domain, first CH domain and first CL domain together form a first Fab, and when combined, the second VH domain, second VL domain, second CHI domain, and second CL domain form a second Fab. In some embodiments, the first VH domain or the second VH domain has at least one mutation relative to a human immunoglobulin VH domain, referred to as the VH mutant residue(s); and the first VL domain or the second VL domain has at least one mutation relative to a human immunoglobulin VL domain, referred to as the VL mutant residue(s).

[0307] In some embodiments, the CHI mutant residue(s) and the CL mutant residue(s) comprise charged amino acids such that the CHI mutant residue(s) and the CL mutant residue(s) comprise a charge pair. In some embodiments, the CHI mutant residue(s) and the CL mutant residue(s) comprise at least two charge pairs. In some embodiments, the charge pairs comprise at least one charge pair on the first CHCL domain and at least one charge pair on the second CHCL domain located at the same positions. A charged pair interaction of amino acid residuescan refer to the electrostatic attraction or repulsion between two amino acids that carry a net positive or negative charge on their side chains or functional groups. For example, a common charged pair interaction is between the amino acids lysine (Lys) and glutamic acid (Glu). Lysine has a positively charged side chain due to the presence of an amino group (NH3+), while glutamic acid has a negatively charged side chain due to the carboxyl group (COO-). These opposite charges create an attractive force between the two amino acids. The charged pair interaction can play a role in protein / protein interactions. The charged pair interaction can attract each other, forming a salt bridge or ionic bond. In protein-protein interactions, complementary charged amino acids can attract each other, facilitating the formation of protein complexes, such as the bispecific binding agents disclosed herein.

[0308] In some embodiments, the CHI mutant residue(s) and the CL mutant residue(s) interact with each other in preference to corresponding non-mutated CHI residue(s) or corresponding non-mutated CL residue(s). In some embodiments, the CHI mutant residue(s) repel a CL domain comprising the corresponding non-mutated CL residue(s) or the CL mutant residue(s) repel a CHI domain comprising the corresponding non-mutated CHI residue(s). In some embodiments, the first VH domain or the second VH domain has at least one mutation relative to a human immunoglobulin VH domain, referred to as the VH mutant residue(s); and the first VL domain or the second VL domain has at least one mutation relative to a human immunoglobulin VL domain, referred to as the VL mutant residue(s). In some embodiments, the VH mutant residue(s) and the VL mutant residue(s) comprise a disulfide bridge pair. A disulfide bridge pair between amino acid residues can refer to the covalent bond formed between two cysteine amino acids through a redox reaction. Cysteine contains a unique sulfur- containing side chain called a thiol group (-SH). Under oxidizing conditions, the thiol groups of two cysteine residues within a protein can undergo oxidation, resulting in the formation of a disulfide bond (S-S) between the two cysteines. This bond creates a covalent link, often referred to as a disulfide bridge. These bridges can affect protein-protein interactions by forming specific intermolecular covalent links between proteins.Amino Acid Modifications

[0309] Alternative compositions and methods of producing multispecific antibodies with correct light chain pairing include various amino acid modifications. For example, Zymeworks describes heterodimers with one or more amino acid modifications in the CHI and / or CL domains, one or more amino acid modifications in the VH and / or VL domains, or a combination thereof, which are part of the interface between the light chain and heavy chain and create preferential pairing between each heavy chain and a desired light chain such thatwhen the two heavy chains and two light chains of the heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains rather than the other (see e.g., W02015181805). Other exemplary methods are described in WO2016026943 (Argen-X), US20150211001, US20140072581A1, US20160039947A1, and US20150368352.Lambda / Kappa Formats

[0310] Multifunctional or multispecific binding agents (e.g., multifunctional or multispecific antibody binding agents) that include the lambda light chain polypeptide and a kappa light chain polypeptides, can be used to allow for heterodimerization. Methods for generating bispecific antibody binding agents comprising the lambda light chain polypeptide and a kappa light chain polypeptides are disclosed in PCT / US17 / 53053 filed on September 22, 2017 and designated publication number WO 2018 / 057955, incorporated herein by reference in its entirety.

[0311] In some embodiments, the multifunctional or multispecific binding agent includes a multispecific antibody binding agent, e.g., an antibody binding agent comprising two binding specificities, e.g., a bispecific antibody binding agent. The multifunctional or multispecific antibody binding agent includes: a lambda light chain polypeptide 1 (LLCP1) specific for a first epitope; a heavy chain polypeptide 1 (HCP1) specific for the first epitope; a kappa light chain polypeptide 2 (KLCP2) specific for a second epitope; and a heavy chain polypeptide 2 (HCP2) specific for the second epitope.

[0312] “Lambda light chain polypeptide 1 (LLCP1)”, as that term is used herein, refers to a polypeptide comprising sufficient light chain (LC) sequence, such that when combined with a cognate heavy chain variable region, can mediate specific binding to its epitope and complex with an HCP1. In some embodiments, it comprises all or a fragment of a CHI region. In some embodiments, an LLCP1 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CHI, or sufficient sequence therefrom to mediate specific binding of its epitope and complex with an HCP1. LLCP1, together with its HCP1, provide specificity for a first epitope (while KLCP2, together with its HCP2, provide specificity for a second epitope). As described elsewhere herein, LLCP1 has a higher affinity for HCP1 than for HCP2.

[0313] “Kappa light chain polypeptide 2 (KLCP2)”, as that term is used herein, refers to a polypeptide comprising sufficient light chain (LC) sequence, such that when combined with a cognate heavy chain variable region, can mediate specific binding to its epitope and complex with an HCP2. In some embodiments, it comprises all or a fragment of a CHI region. In someembodiments, a KLCP2 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CHI, or sufficient sequence therefrom to mediate specific binding of its epitope and complex with an HCP2. KLCP2, together with its HCP2, provide specificity for a second epitope (while LLCP1, together with its HCP1, provide specificity for a first epitope).

[0314] “Heavy chain polypeptide 1 (HCP1)”, as that term is used herein, refers to a polypeptide comprising sufficient heavy chain (HC) sequence, e.g., HC variable region sequence, such that when combined with a cognate LLCP1, can mediate specific binding to its epitope and complex with an HCP1. In some embodiments, it comprises all or a fragment of a CHlregion. In some embodiments, it comprises all or a fragment of a CH2 and / or CH3 region. In some embodiments, an HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CHI, CH2, and CH3, or sufficient sequence therefrom to: (i) mediate specific binding of its epitope and complex with an LLCP1, (ii) to complex preferentially, as described herein to LLCP1 as opposed to KLCP2; and (iii) to complex preferentially, as described herein, to an HCP2, as opposed to another molecule of HCP1. HCP1, together with its LLCP1, provide specificity for a first epitope (while KLCP2, together with its HCP2, provide specificity for a second epitope).

[0315] “Heavy chain polypeptide 2 (HCP2)”, as that term is used herein, refers to a polypeptide comprising sufficient heavy chain (HC) sequence, e.g., HC variable region sequence, such that when combined with a cognate LLCP1, can mediate specific binding to its epitope and complex with an HCP1. In some embodiments, it comprises all or a fragment of a CHlregion. In some embodiments, it comprises all or a fragment of a CH2 and / or CH3 region. In some embodiments, an HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CHI, CH2, and CH3, or sufficient sequence therefrom to: (i) mediate specific binding of its epitope and complex with an KLCP2, (ii) to complex preferentially, as described herein to KLCP2 as opposed to LLCP1; and (iii) to complex preferentially, as described herein, to an HCP1, as opposed to another molecule of HCP2. HCP2, together with its KLCP2, provide specificity for a second epitope (while LLCP1, together with its HCP1, provide specificity for a first epitope).

[0316] In some embodiments, in the multifunctional polypeptide binding agent as described herein:LLCP1 has a higher affinity for HCP1 than for HCP2; and / or KLCP2 has a higher affinity for HCP2 than for HCP1.

[0317] In some embodiments, the affinity of LLCP1 for HCP1 is sufficiently greater than its affinity for HCP2, such that under preselected conditions, e.g., in aqueous buffer, e.g., at pH7, in saline, e.g., at pH 7, or under physiological conditions, at least 75, 80, 90, 95, 98, 99, 99.5, or 99.9 % of the multifunctional or multispecific antibody binding agent molecules have a LLCP1 complexed, or interfaced with, a HCP1.

[0318] In some embodiments, in the multifunctional polypeptide binding agent as described herein: the HCP1 has a greater affinity for HCP2, than for a second molecule of HCP1; and / or the HCP2 has a greater affinity for HCP1, than for a second molecule of HCP2.

[0319] In some embodiments, the affinity of HCP1 for HCP2 is sufficiently greater than its affinity for a second molecule of HCP1, such that under preselected conditions, e.g., in aqueous buffer, e.g., at pH 7, in saline, e.g., at pH 7, or under physiological conditions, at least 75%, 80, 90, 95, 98, 99 99.5 or 99.9 % of the multifunctional or multispecific antibody binding agent molecules have a HCP1 complexed, or interfaced with, a HCP2.

[0320] In another aspect, described herein is a method for making, or producing, a multifunctional or multispecific antibody binding agent. The method includes: (i) providing a first heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three or all of a first heavy chain variable region (first VH), a first CHI, a first heavy chain constant region (e.g., a first CH2, a first CH3, or both)); (ii) providing a second heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three or all of a second heavy chain variable region (second VH), a second CHI, a second heavy chain constant region (e.g., a second CH2, a second CH3, or both)); (iii) providing a lambda chain polypeptide (e.g., a lambda light variable region (VIA), a lambda light constant chain (VIA), or both) that preferentially associates with the first heavy chain polypeptide (e.g., the first VH); and (iv) providing a kappa chain polypeptide (e.g., a lambda light variable region (VIA), a lambda light constant chain (VIA), or both) that preferentially associates with the second heavy chain polypeptide (e.g., the second VH), under conditions where (i)-(iv) associate.

[0321] In some embodiments, the first and second heavy chain polypeptides form an Fc interface that enhances heterodimerization.

[0322] In some embodiments, (i)-(iv) (e.g., nucleic acid encoding (i)-(iv)) are introduced in a single cell, e.g., a single mammalian cell, e.g., a CHO cell. In some embodiments, (i)-(iv) are expressed in the cell. In some embodiments, (i)-(iv) (e.g., nucleic acid encoding (i)-(iv)) are introduced in different cells, e.g., different mammalian cells, e.g., two or more CHO cell. In some embodiments, (i)-(iv) are expressed in the cells.

[0323] In some embodiments, the method further comprises purifying a cell-expressed antibody binding agent, e.g., using a lambda- and / or- kappa-specific purification, e.g., affinity chromatography.

[0324] In some embodiments, the method further comprises evaluating the cell-expressed multifunctional or multispecific antibody binding agent. For example, the purified cell- expressed multifunctional or multispecific antibody binding agent can be analyzed by techniques known in the art, include mass spectrometry. In some embodiments, the purified cell-expressed antibody binding agent is cleaved, e.g., digested with papain to yield the Fab moieties and evaluated using mass spectrometry.

[0325] In some embodiments, the method produces correctly paired kappa / lambda multifunctional or multispecific, e.g., bispecific, antibody binding agents in a high yield, e.g., at least 75%, 80, 90, 95, 98, 99 99.5 or 99.9 %.

[0326] In other embodiments, the multifunctional or multispecific, e.g., a bispecific, antibody binding agent that includes: (i) a first heavy chain polypeptide (HCP1) (e.g., a heavy chain polypeptide comprising one, two, three or all of a first heavy chain variable region (first VH), a first CHI, a first heavy chain constant region (e.g., a first CH2, a first CH3, or both)), e.g., wherein the HCP1 binds to a first epitope; (ii) a second heavy chain polypeptide (HCP2) (e.g., a heavy chain polypeptide comprising one, two, three or all of a second heavy chain variable region (second VH), a second CHI, a second heavy chain constant region (e.g., a second CH2, a second CH3, or both)), e.g., wherein the HCP2 binds to a second epitope; (iii) a lambda light chain polypeptide (LLCP 1) (e.g., a lambda light variable region (VIA), a lambda light constant chain (VIA), or both) that preferentially associates with the first heavy chain polypeptide (e.g., the first VH), e.g., wherein the LLCP1 binds to a first epitope; and (iv) a kappa light chain polypeptide (KLCP2) (e.g., a kappa light variable region (VLK), a kappa light constant chain (VLK), or both) that preferentially associates with the second heavy chain polypeptide (e.g., the second VH), e.g., wherein the KLCP2 binds to a second epitope.

[0327] In some embodiments, the first and second heavy chain polypeptides form an Fc interface that enhances heterodimerization. In some embodiments, the multifunctional or multispecific antibody binding agent has a first binding specificity that includes a hybrid VLk- CL heterodimerized to a first heavy chain variable region connected to the Fc constant, CH2- CH3 domain (having a knob modification) and a second binding specificity that includes a hybrid VLK-CLK heterodimerized to a second heavy chain variable region connected to the Fc constant, CH2-CH3 domain (having a hole modification).Nucleic Acid Molecules

[0328] Provided herein, are methods of making the binding agents of the present disclosure, including recombinant polynucleotide molecules, vectors comprising the recombinant polynucleotide molecules, and cells comprising the recombinant polynucleotide molecules. Binding agents of the present disclosure are synthesized using the techniques of recombinant DNA and protein expression. For example, for the synthesis of DNA encoding a dual IgG of the disclosure, suitable DNA sequences encoding the constant domains of the heavy and light chains are widely available.

[0329] In some embodiments, “nucleic acid” or “polynucleotide” can refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0330] Described herein, in certain embodiments, is an isolated nucleic acid molecule comprising a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% sequence identity to the nucleotide sequence encoding the multifunctional polypeptide molecule as described herein. Nucleic acids encoding the aforementioned binding agents are also disclosed.

[0331] In certain embodiments, the invention features nucleic acids comprising nucleotide sequences that encode heavy and light chain variable regions and CDRs or hypervariable loops of the antibody binding agents, as described herein. For example, the invention features a first and second nucleic acid encoding heavy and light chain variable regions, respectively, of an antibody binding agent chosen from one or more of the antibody binding agents as described herein.

[0332] Provided herein, are recombinant polynucleotide molecule comprising the polynucleotide sequences encoding the binding agent of the present disclosure. In some embodiments, encoding can refer to the inherent property of specific sequences of nucleotidesin a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. For example, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. In some cases, the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. In some embodiments, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain one or more introns.

[0333] In some embodiments, the recombinant polynucleotide molecule is an isolated recombinant polynucleotide molecule. Sequences encoding the selected variable domains are inserted by standard methods, and the resulting nucleic acids encoding full-length heavy and light chains are transduced into suitable host cells and expressed. Alternatively, the nucleic acids can be expressed in a cell-free expression system, which can provide more control over oxidation and reduction conditions, pH, folding, glycosylation, and the like.Vectors

[0334] Described herein, in certain embodiments, is a vector comprising one or more of the nucleic acid molecules as described herein.

[0335] Further provided herein are vectors comprising the nucleotide sequences encoding antibody binding agents, bispecific binding agents, and / or a multispecific or multifunctional binding agent described herein. In some embodiments, the vectors comprise nucleic acid sequences encoding antibody binding agents, or multispecific or multifunctional binding agent described herein. In some embodiments, the vectors comprise the nucleotide sequences described herein. The vectors include, but are not limited to, a virus, plasmid, cosmid, lambda phage or a yeast artificial chromosome (YAC).

[0336] Numerous vector systems can be employed. For example, one class of vectors utilizes DNA elements which are derived from animal viruses such as, for example, bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (Rous Sarcoma Virus, MMTV or MOMLV) or SV40 virus. Another class of vectors utilizes RNAelements derived from RNA viruses such as Semliki Forest virus, Eastern Equine Encephalitis virus and Flaviviruses.

[0337] Additionally, cells which have stably integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow for the selection of transfected host cells. The marker may provide, for example, prototropy to an auxotrophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper, or the like. The selectable marker gene can be either directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals.

[0338] Once the expression vector or DNA sequence containing the constructs has been prepared for expression, the expression vectors may be transfected or introduced into an appropriate host cell. Various techniques may be employed to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid based transfection or other conventional techniques. In the case of protoplast fusion, the cells are grown in media and screened for the appropriate activity.

[0339] Methods and conditions for culturing the resulting transfected cells and for recovering the antibody binding agent produced are known to those skilled in the art, and may be varied or optimized depending upon the specific expression vector and mammalian host cell employed, based upon the present description.

[0340] Provided herein, are vectors comprising the recombinant polynucleotide molecule of the present disclosure. In one aspect, the binding agents of the present disclosure relate to nucleic acid molecules comprising nucleotide sequences encoding the binding agents of the disclosure, including expression cassettes, and expression vectors containing these nucleic acid molecules operably linked to heterologous nucleic acid sequences such as, for example, regulatory sequences which direct in vivo expression of the protein in a host cell.

[0341] In some embodiments, a vector, a plasmid, or a virus contains one or more of the nucleic acid molecules encoding any binding agent disclosed herein. In some embodiments, a vector, a plasmid, or a virus contains one or more of the nucleic acid molecules encoding four of the heavy / light chains of the binding agent disclosed herein (e.g., one vector may contain nucleic acid molecules encoding both a heavy chain and a light chain of the first binding domain of the binding agent disclosed herein as wells as a heavy chain and a light chain of the second binding domain of the binding agent disclosed herein). The nucleic acid molecules canbe contained within a vector that is capable of directing their expression in, for example, a cell that has been transformed / transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or readily prepared by a skilled artisan. See for example, Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY : Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley -Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0342] Methods disclosed herein may include collecting the recombinant polynucleotide molecule described herein. Methods disclosed herein may include collecting the recombinant polynucleotide described herein. Methods disclosed herein may include collecting the polypeptide (e.g., bispecific binding agent) described herein. In some embodiments, the collecting comprises lysing a cell. A cell may be lysed via high pressure, osmotic shock or pressure, low temperature, sonication, homogenization, or a combination thereof. The resulting solution, or lysate of the cell, may be collected and subjected to downstream analysis or processing such as purification. In some embodiments, the collecting the polypeptide comprises purifying the polypeptide. In some embodiments, purifying does not comprise lysing the cell. In some embodiments, the polypeptide may be secreted. The secretion of the polypeptide may allow the polypeptide to be collected in the media. The secretion of the polypeptide may allow the polypeptide to be collected without lysis of the cell.

[0343] The recombinant polynucleotide molecule may be purified, or otherwise concentrated or isolated such a solution contains predominantly the recombinant polynucleotide molecule. In some embodiments, the purification is performed on the soluble fraction of the cell lysate. In some embodiments, the soluble fraction of the cell lysate is obtained by the centrifugation of cell lysate and collecting the supernatant. In some embodiments, the purification is performed on the insoluble fraction of the cell lysate. In some embodiments, the insoluble fraction of the cell lysate is obtained by the centrifugation of cell lysate and collecting the pellet formed. In some embodiments, the insoluble fraction comprises inclusion bodies that include the recombinant polynucleotide molecule. In some embodiments, the purifying comprises isolating the polypeptide is purified from the inclusion bodies. In some embodiments, purifying comprises isolating from inclusion bodies. Isolating the polypeptide from the inclusion bodies may comprise the use of denaturants and chaotropes, such as guanidine or urea, to solubilize the inclusion bodies. In some embodiments, the purifying uses an agent that specifically binds the polypeptide. For example, the agent may be protein with a binding affinity to the polypeptide. In some embodiment, the agent that specifically binds the polypeptide is immobilized on a solid support such as a bead and is used to capture the polypeptide.

[0344] In some embodiments, purifying comprises using a chromatography. Chromatography may be performed by using the physical characteristics of the polypeptide, such as charge, shape, or polarity, to separate the polypeptide from other protein. In some embodiments, the chromatography comprises ion exchange chromatography, size exclusion chromatography, gel chromatography, reverse phase chromatography, affinity chromatography, or a combination thereof. Multiple forms of chromatography may be used sequentially to increase the purity of the polypeptide. Chromatography may be performed by introducing the cell lysate or polypeptide containing solution into a column containing resin or other solid supports. The resin may interact different with different proteins and polypeptides resulting in a separation of the proteins based on physical characteristics. For example, the resin may contain nickel and may interact with a His tag on the polypeptide. In some embodiments, the chromatography is selected from the group consisting of an ion exchange chromatography, a size exclusion chromatography, a reverse phase chromatography, and an affinity chromatography. In some embodiments, purifying comprises using an agent that specifically binds the polypeptide. In some cases, the tag or affinity tag of the polypeptide as described herein can be used for the purification of the polypeptide as described herein.

[0345] In some embodiments, purifying comprises using a dialysis. Dialysis can be used as a method for purification of the polypeptides disclosed herein. Dialysis can involve the separation of the polypeptides disclosed herein from smaller molecules using a semipermeable membrane. Dialysis takes advantage of the principle of diffusion, where molecules move from an area of higher concentration to an area of lower concentration. In some embodiments, the dialysis is performed using a dialysis membrane. In some embodiments, the dialysis is performed using a dialysis tubing. A dialysis membrane and / or a dialysis tubing can be a semipermeable membrane with specific molecular weight cutoff (MWCO). The MWCO can determine the size of molecules that can pass through the semipermeable membrane. For example, the MWCO can allow for sugar moieties to pass through the semipermeable membrane, but not the polypeptides disclosed herein.

[0346] The methods as disclosed elsewhere herein may yield a particular polypeptide yield. In some embodiments, the methods as disclosed elsewhere herein produce polypeptide at an amount of at least 0.1 mgs(milligrams) per liter(L) of media. In some embodiments, the methods produce polypeptide at an amount of at least 1 mg per liter of media. In some embodiments, the methods produce polypeptide at an amount of at least 5 mgs per liter of media. In some embodiments, the methods produce polypeptide at an amount of at least 10 mgs per liter of media. In some embodiments, the methods produce polypeptide at an amount of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more mgs per liter of media.Cells

[0347] Described herein, in certain embodiments, is a cell comprising the nucleic acid as described herein or the vector as described herein.

[0348] In another aspect, described herein are host cells and vectors containing the nucleic acids. The nucleic acids may be present in a single vector or separate vectors present in the same host cell or separate host cell. The host cell can be a eukaryotic cell, e.g., a mammalian cell, an insect cell, a yeast cell, or a prokaryotic cell, e.g., E. coli. For example, the mammalian cell can be a cultured cell or a cell line. Exemplary mammalian cells include lymphocytic cell lines (e.g., NSO), Chinese hamster ovary cells (CHO), COS cells, oocyte cells, and cells from a transgenic animal, e.g., mammary epithelial cell.

[0349] In some embodiments, described herein are host cells comprising a nucleic acid encoding an antibody binding agent as described herein.

[0350] In some embodiments, described herein are the host cells genetically engineered to comprise nucleic acids encoding the antibody binding agent.

[0351] In some embodiments, the host cells are genetically engineered by using an expression cassette. The phrase “expression cassette,” refers to nucleotide sequences, which are capable of affecting expression of a gene in hosts compatible with such sequences. Such cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in effecting expression may also be used, such as, for example, an inducible promoter.

[0352] In some embodiments, described herein are host cells comprising the vectors described herein. The cell can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0353] In some embodiments, the methods described can be performed in a host cell, or in vitro, in cell-free synthetic systems. Host cells may be any that can be robustly recoded. These can be bacterial cells that have well developed genetic systems, of which E. coli is exemplary. Other bacterial species can also be used. In some embodiments, cell-free systems for producing the proteins may be coupled transcription / translation systems or only translation systems. Notably, in some embodiments, biological syntheses are utilized rather than chemical synthesis.

[0354] In one aspect the present disclosure provides a cell comprising the recombinant polynucleotide molecule disclosed herein. Culturing of recoded cells with the constructed nucleic acid sequences may be by any means known in the art. In some embodiments, the culturing may be batch or continuous, in shaker flasks or in fermenters or immobilized on solid surfaces, such as small particles contained in larger vessels.

[0355] In some embodiments, the cell can be a plurality of cells. In some cases, the plurality of cells can be from about 1 cell to about 1 billion cells. In some cases, the plurality of cells can be about 0 cells, about 10 cells, about 100 cells, about 1,000 cells, about 10,000 cells, about 100,000 cells, about 1,000,000 cells, about 10,000,000 cells, about 100,000,000 cells, about 1,000,000,000 cells. In some cases, the plurality of cells can be at least about 0 cells, about at least 10 cells, about at least 100 cells, about at least 1,000 cells, about at least 10,000 cells, about at least 100,000 cells, about at least 1,000,000 cells, about at least 10,000,000 cells, about at least 100,000,000 cells, about at least 1,000,000,000 cells, or more.

[0356] In some embodiments, the cell can be a bacterial cell. In some cases, the bacterial cell can be a bio-contained strain. In some cases, the bacterial cell can be a multi-virus resistance bacterial cell. In some embodiments, the bacterial cell can be from a bacterial genus. Non-limiting examples of bacterial geneses include Staphylococcus, Streptococcus, Enterococcus, Moraxella, Neisseria, Corynebacterium, Bacillus, Lactobacillus, Listeria, Citrobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Serratia, Hafinia, Morganella, Providencia, Salmonella, Shigella, Yersinia, Acinetobacter, Pseudomonas, Strenotrophomonas, Burkholderia, Haemophilus, Legionella, Achromobacter, Aeromonas, Alcaligenes, Campylobacter, Flavobacterium, Helicobacter, Pasteurella, Bacteroides, Clostridium, Propionibacterium, Prevotella, Mycobacterium, Mycoplasma, Actinomyces, Acetobacter, Bordetella, Vibrio, and Nocardia. In some embodiments, the bacterial cell can be a bacterial species. Non-limiting examples of bacterial species include Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Moraxella catharralis, Neisseria meningitidis, Listeria monocytogenes, Acetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus mycoides, Bacillus subtilis, Bacteroides fragilis, Bacteroides gingivalis, Bordetella bronchiseptica, Bordetella pertussis, Burkholderia mallei, Burkholderiahosphora, Campylobacter jejuni, Campylobacter pylori, Clostridium botulinum, Clostridium difficule, Corynebacterium diphtheria Corynebacterium fusiforme, Enterococcus avium, Enterococcus durans, Enterococcus gallinarum, Enterococcus maloratus, Haemophilus influenzae, Haemophilus pertussis, Haemophilus parainfluenzae, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus lactis, Legionella pneumophila, Mycobacterium avium, Mycobacterium bovis, Mycoplasma hominis, Mycoplasma fermentans, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tulanensis, Prevotella melaninogenica, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhi, Vibrio comma, Vibrio vulnificus, and Yersinia enterocolitica.

[0357] In some embodiments, the cell can be a mammalian cell. In some embodiments, the cell can be a mammalian cell line. Non-limiting examples of mammalian cell lines include HeLa cells, HEK293 cells, CHO cells, NIH / 3T3 cells, Jurkat cells, RAW 264.7 cells, SH- SY5Y cells, MCF-7 cells, A549 cells, and U87 cells.IILPHARMACEUTICAL COMPOSITION

[0358] Provided herein is a pharmaceutical composition, comprising the bispecific binding agent disclosed herein, and a pharmaceutically acceptable excipient. For example, binding agents of the disclosure can be administered using formulations used for administering antibodies and antibody-based therapeutics, or formulations based thereon. In some embodiments, provided herein is a pharmaceutical composition comprising: (i) any binding agent described herein; and (ii) a pharmaceutically acceptable carrier, excipient, or additive. The pharmaceutical compositions described herein can provide homogenous protein therapeutics for disease and conditions for which homogeneity of glycosylation of the therapeutic protein(s) is crucial for treatment.

[0359] In some embodiments, the pharmaceutical composition of the present disclosure may contain pharmaceutically acceptable carriers, excipients, or additives depending on the route of administration. In some embodiments, carriers, excipients, or additives may include, but are not limited to, water (e.g. , sterile water), saline, buffered saline, dextrose, glycerol, ethanol, sterile isotonic aqueous buffer, binding agent, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like, and combinations thereof. In some embodiments, the at least one pharmaceutically acceptable carrier, excipient, or additive comprises saline or sterile water. Additives used are chosen from, but not limited to, the above or combinations thereof depending on the dosage form.

[0360] In some embodiments, the pharmaceutical composition is formulated for oral administration, intravenous injection, intradermal injection, subcutaneous injection, intrathecal administration, intracerebral administration, intracerebroventricular injection, topical administration, inhalation, or nasal administration. In some embodiments, the pharmaceutical composition is formulated for transdermal administration (which may include a penetration enhancement agent), buccal administration, or suppository administration. In some embodiments, the pharmaceutical composition of the present disclosure may be administered, either orally or parenterally, systemically or locally. For example, intravenous injection such as drip infusion, intramuscular injection, intrapleural injection, intraperitoneal injection,subcutaneous injection, suppositories, intestinal lavage, oral enteric coated tablets, and the like can be selected, and the method of administration may be chosen, as appropriate, depending on the age and the condition of the subject.

[0361] In some embodiments, the pharmaceutical composition is a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spray solution, a suppository, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, an ointment, a lotion, an eye drop, or an ear drop.

[0362] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a drug, a compound, a small molecule, a macrocycle, an antibody or fragment thereof, a polynucleotide, a peptide, a payload, or a protein. In some embodiments, the therapeutic agent is synthetic or biosynthetic. In some embodiments, the additional therapeutic agent reduces expression of an efflux transporter or inhibits an efflux transporter. In some embodiments, the additional therapeutic agent increases expression of an influx transporter or promotes activity of an influx transporter.

[0363] In some embodiments, drugs or payloads are selected from the group consisting of DM1 (maytansine, N2'-deacetyl-N2'-(3 -mercapto- 1 -oxopropyl)- or N2'-deacetyl-N2'-(3- mercapto-l-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproyl- monomethylauristatin-D or N- methyl-L-valyl-N-[(l S,2R)-2-methoxy-4-[(2S)-2-[(lR,2R)-l- methoxy-2-methyl-3 -oxo-3 -[(1 S)-2 -phenyl- 1 -(2-thiazolyl)ethyl]amino]propyl]- 1 - pyrrolidinyl]-l-[(lS)-l-methylpropyl]-4-oxobutyl]-N-methyl-(9CI)-L-valinamide), mc- MMAF (maleimidocaproyl-monomethylauristatin F or N-[6-(2,5-dihydro-2,5-dioxo-lH- pyrrol-l-yl)-l-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4- (methylamino)heptanoyl-(aR,pR,2S)-p-methoxy-a-methyl-2-pyrrolidinepropanoyl-L- phenylalanine) and mc-Val-Cit-PABA-MMAE (6-maleimidocaproyl-ValcCit-(p- aminobenzyloxy carbonyl)- monomethylauristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo- 1 H-pyrrol-l-yl)-l-oxohexyl]-L-valyl-N5-(aminocarbonyl)-L- ornithyl]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-N-[(lS,2R)-4-[(2S)-2- [(lR,2R)-3-[[(lR,2S)-2-hydroxy-l-methyl-2-phenylethyl]amino]-l-methoxy-2-methyl-3- oxopropyl]-l-pyrrolidinyl]-2-methoxy-l-[(lS)-l-methylpropyl]-4-oxobutyl]-N-methyl-L- valinamide). DM1 is a derivative of the tubulin inhibitor maytansine while monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF) are auristatin derivatives. The preferred payloads of the present disclosure are selected from the group consisting of mc-MMAF and mc-Val-Cit- PABA-MMAE.

[0364] The herein described pharmaceutical compositions may be formulated into different forms; for example, they may be formulated into a solid form (e.g., powders, tablets, capsule, creams, films, granules, paste, and gels) or a liquid form (e.g., suspension, solution, and syrup). In embodiments, the pharmaceutical composition is formulated into a tablet, a capsule, a powder, which tablet, capsule or powder may or may not have sustained release characteristics.

[0365] In embodiments, for solid dosage forms used in oral, the active ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients, or additives, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents, in the case of capsules, tablets, and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type can also be prepared using fillers in soft and hard-filled gelatin capsules, and excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more binding agents of the disclosure, and for example, at a concentration of 25%-75%.

[0366] In some embodiments, the pharmaceutical composition is in a powder form. In embodiments, the pharmaceutical composition is a dispersible powder. The pharmaceutically acceptable carrier, excipient, or additive suitable for formulating a dispersible powder includes, without limitation, sodium chloride, sugars (e.g., sucrose, lactose, trehalose, and mannitol), and derived calcium or other divalent cations (e.g., those obtained from calcium chloride, zinc chloride, manganese chloride and magnesium chloride). In embodiments, the dispersible powder has a mean particle size from about 0.2 to 50 pm, 0.2 to 25 pm, 0.2 to 15 pm, 0.2 to 10 pm, 0.5 to 10 pm, 1 to 10 pm, 1 to 8 pm, 1 to 6 pm, 1 to 5 pm, 1 to 4 pm, 1 to 3 pm, 2 to 10 pm, 2 to 8 pm, 2 to 6 pm, or 2 to 4 pm.

[0367] In some embodiments, the pharmaceutical composition is prepared by a method comprising (a) spray-drying a liquid composition comprising one or more binding agents and the at least one pharmaceutically acceptable carrier, excipient, or additive, and (b) collecting the spray-dried product of step (a) as a dispersible powder containing one or more binding agents, functional fragment thereof, or variant thereof. In embodiments, the liquid composition comprises a carrier such as saline, water, or alcohol. In embodiments, the liquid composition comprises sterile water. In some embodiments, the liquid composition comprises sodium chloride. In some embodiments, the liquid composition comprises a sugar.

[0368] In some embodiments, the liquid composition has a concentration of the binding agent of about from 0.1 mg / ml to 400 mg / ml, 1 mg / ml to 300 mg / ml, 1 mg / ml to 200 mg / ml, 1 mg / ml to 100 mg / ml, 1 mg / ml to 80 mg / ml, 1 mg / ml to 50 mg / ml, 1 mg / ml to 25 mg / ml, 5 mg / ml to 100 mg / ml, 5 mg / ml to 50 mg / ml, or 5 mg / ml to 25 mg / ml. In some embodiments, the liquid composition has a concentration of the binding agent of about from 1 mg / ml to 80 mg / ml. In some embodiments, the binding agent is present in the dispersible powder in an amount of about 1% to 100% by weight. In some embodiments, the binding agent is present in the dispersible powder in an amount of about at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% by weight. In some embodiments, the binding agent is present in the dispersible powder in an amount of about at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% by weight. In some embodiments, the dispersible powder comprises a sugar in an amount of about from 10% to 90% by weight. In some embodiments, the dispersible powder comprises sodium chloride in an amount of about from 10% to 90% by weight.

[0369] In some embodiments, the pharmaceutical composition is prepared by a method further comprising combining one or more additional therapeutics with one or more binding agents. For example, in some embodiments, the additional therapeutic may be combined with the dispersible powder that contains one or more binding agents. For another example, in some embodiments, the additional therapeutic may be combined with one or more binding agents to produce the liquid composition. For yet another example, in some embodiments, the additional therapeutic may be added into the liquid composition that comprises one or more binding agents.

[0370] In some embodiments, the pharmaceutical composition is in a liquid form such as suspension, solution, and syrup, which liquid form may or may not have a sustained release feature. In some embodiments, the pharmaceutical composition is an aerosolizable solution or suspension. Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the active ingredient(s) together with conventional pharmaceutically-acceptablecarriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

[0371] A suitable carrier for the aerosolable solution or suspension may comprise, for example, saline, water, alcohol (e.g., ethanol), or a combination thereof. In embodiments, the aerosolable solution or suspension comprises saline or sterile water. In certain embodiments, the aerosolable solution or suspension has a concentration of the binding agent, functional fragment thereof, or variant thereof of about from 0.1 mg / mL to 50 mg / mL, 0.1 mg / mL to 20 mg / mL, 0.1 mg / mL to 10 mg / mL, 0.5 mg / mL to 10 mg / mL, 0.5 mg / mL to 5 mg / mL, 0.5 mg / mL to 2 mg / mL, or 0.75 mg / mL to 1.25 mg / mL. In embodiments, the aerosolable solution or suspension has a concentration of the binding agent, functional fragment thereof, or variant thereof of about at least 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 1.25 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL. In embodiments, the aerosolable solution or suspension has a concentration of the binding agent, functional fragment thereof, or variant thereof of about at most 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 1.25 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, or 50 mg / mL.

[0372] In some embodiments, the pharmaceutical composition is a dosage form and comprises 0.1 to 50 mL, 0.1 to 20 mL, 0.1 to 10 mL, 0.5 to 20 mL, 0.5 to 10 mL, 0.5 to 5 mL, 0.5 to 4 mL, 0.5 to 3 mL, 0.5 to 2 mL, 0.5 to 1.5 mL, 0.5 to 1 mL, 0.75 to 5 mL, 0.75 to 4 mL, 0.75 to 3 mL, 0.75 to 2.5 mL, 0.75 to 2 mL, 0.75 to 1.5 mL, 0.75 to 1.25 mL, or 0.75 to 1 mL. In some embodiments, the pharmaceutical composition is a dosage form and comprises at least about 0.1 mL, 0.25 mL, 0.5 mL, 0.75 mL, 1 mL, 1.25 mL, 1.5 mL, 1.75 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, or 20 mL. In some embodiments, the pharmaceutical composition is a dosage form and comprises at most about 0.5 mL, 0.75 mL, 1 mL, 1.25 mL, 1.5 mL, 1.75 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, or 50 mL.

[0373] In some embodiments, the pharmaceutical composition is a dosage form and the binding agent is present in the dosage form at an amount of about 0.1 mg to 20 mg, 0.1 mg to 10 mg, 0.1 mg to 5 mg, 0.5 mg to 4 mg, 0.5 mg to 3 mg, 0.5 mg to 2.5 mg, 0.5 mg to 2 mg, 0.5 mg to 1.5 mg, 0.75 mg to 1.5 mg, 0.75 mg to 1.25 mg, or 1.5 mg to 2.5 mg. In some embodiments, the pharmaceutical composition is a dosage form and the binding agent ispresent in the dosage form at an amount of at least about 0.1 mg, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, or 20 mg. In some embodiments, the pharmaceutical composition is a dosage form and the binding agent is present in the dosage form at an amount of at most 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, or 50 mg.

[0374] In some embodiments, a weight ratio of the binding agent and the additional therapeutic present in the pharmaceutical composition for an administration is at least 1 : 1, 2: 1, 3:1, 4:1, 5:1, 6:1, 10:1, 25:1, 50:1, 100: 1, 200:1, 300:1, 400:1, 500:1, 600:1, 800:1, 6000:1, or 60000:1. In some embodiments, a weight ratio of the binding agent and the additional therapeutic present in the pharmaceutical composition for an administration is at most 1 : 1, 2: 1, 3:1, 4:1, 5:1, 6:1, 10:1, 25:1, 50:1, 100: 1, 200:1, 300:1, 400:1, 500:1, 600:1, 800:1, 6000:1, or 60000:1. In some embodiments, a weight ratio of the binding agent and the additional therapeutic present in the pharmaceutical composition for an administration is about from 200:1 to 1000:1, 400:1 to 800:1, or 500:1 to 700:1.

[0375] In some embodiments, a weight ratio of the additional therapeutic and the binding agent present in the pharmaceutical composition for an administration is at least 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 10:1, 25:1, 50:1, 100: 1, 200:1, 300:1, 400:1, 500:1, 600:1, 800:1, 6000:1, or 60000:1. In some embodiments, a weight ratio of the additional therapeutic and the binding agent present in the pharmaceutical composition for an administration is at most 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 10:1, 25:1, 50:1, 100: 1, 200:1, 300:1, 400:1, 500:1, 600:1, 800:1, 6000:1, or 60000:1. In some embodiments, a weight ratio of the additional therapeutic and the binding agent present in the pharmaceutical composition for an administration is about from 200:1 to 1000:1, 400:1 to 800:1, or 500:1 to 700:1.IV.METHODS OF TREATING A DISORDER

[0376] Provided herein is a method of treating a disorder and / or condition in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective amount of the binding agent disclosed herein. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein.

[0377] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a variety of mammalian species utilized in research for various purposes, including medical, biological, and veterinary investigations. In some embodiments, the mammal is a mouse, a rat, a guinea pig, a rabbit, a hamster, a ferret, a cat, a pig, a dog, or a non-human primate. In someembodiments, the non-human primate can be rhesus macaques, marmosets, and / or chimpanzees. In some embodiments, the subject is a human.

[0378] In some cases, the subjects may be at least about 2 years old, at least about 3 years old, at least about 4 years old, at least about 5 years old, at least about 6 years old, at least about 7 years old, at least about 8 years old, at least about 9 years old, at least about 10 years old, at least about 11 years old, at least about 12 years old, at least about 13 years old, at least about 14 years old, at least about 15 years old, at least about 16 years old, at least about 17 years old, at least about 18 years old, at least about 19 years old, at least about 20 years old, at least about 21 years old, at least about 22 years old, at least about 23 years old, at least about 24 years old, at least about 25 years old, at least about 26 years old, at least about 27 years old, at least about 28 years old, at least about 29 years old, at least about 30 years old, at least about 31 years old, at least about 32 years old, at least about 33 years old, at least about 34 years old, at least about 35 years old, at least about 36 years old, at least about 37 years old, at least about 38 years old, at least about 39 years old, at least about 40 years old, at least about 41 years old, at least about 42 years old, at least about 43 years old, at least about 44 years old, at least about 45 years old, at least about 46 years old, at least about 47 years old, at least about 48 years old, at least about 49 years old, at least about 50 years old, at least about 51 years old, at least about 52 years old, at least about 53 years old, at least about 54 years old, at least about 55 years old, at least about 56 years old, at least about 57 years old, at least about 58 years old, at least about 59 years old, at least about 60 years old, at least about 61 years old, at least about 62 years old, at least about 63 years old, at least about 64 years old, at least about 65 years old, at least about 66 years old, at least about 67 years old, at least about 68 years old, at least about 69 years old, at least about 70 years old, at least about 71 years old, at least about 72 years old, at least about 73 years old, at least about 74 years old, at least about 75 years old, at least about 76 years old, at least about 77 years old, at least about 78 years old, at least about 79 years old, at least about 80 years old, at least about 81 years old, at least about 82 years old, at least about 83 years old, at least about 84 years old, at least about 85 years old, at least about 86 years old, at least about 87 years old, at least about 88 years old, at least about 89 years old, at least about 90 years old, at least about 91 years old, at least about 92 years old, at least about 93 years old, at least about 94 years old, at least about 95 years old, at least about 96 years old, at least about 97 years old, at least about 98 years old, at least about 99 years old, or at least about 100 years old. In some cases, the subjects may be at most about 2 years old, at most about 3 years old, at most about 4 years old, at most about 5 years old, at most about 6 years old, at most about 7 years old, at most about 8 years old, at most about 9 years old, at most about 10 years old, at most about 11 years old, atmost about 12 years old, at most about 13 years old, at most about 14 years old, at most about 15 years old, at most about 16 years old, at most about 17 years old, at most about 18 years old, at most about 19 years old, at most about 20 years old, at most about 21 years old, at most about 22 years old, at most about 23 years old, at most about 24 years old, at most about 25 years old, at most about 26 years old, at most about 27 years old, at most about 28 years old, at most about 29 years old, at most about 30 years old, at most about 31 years old, at most about 32 years old, at most about 33 years old, at most about 34 years old, at most about 35 years old, at most about 36 years old, at most about 37 years old, at most about 38 years old, at most about 39 years old, at most about 40 years old, at most about 41 years old, at most about 42 years old, at most about 43 years old, at most about 44 years old, at most about 45 years old, at most about 46 years old, at most about 47 years old, at most about 48 years old, at most about 49 years old, at most about 50 years old, at most about 51 years old, at most about 52 years old, at most about 53 years old, at most about 54 years old, at most about 55 years old, at most about 56 years old, at most about 57 years old, at most about 58 years old, at most about 59 years old, at most about 60 years old, at most about 61 years old, at most about 62 years old, at most about 63 years old, at most about 64 years old, at most about 65 years old, at most about 66 years old, at most about 67 years old, at most about 68 ye...

Claims

CLAIMS1. A method of degrading an extracellular soluble target protein, the method comprising: contacting the extracellular soluble target protein and a membrane-associated protein with a binding agent, wherein the binding agent comprises:(i) a first binding domain that specifically binds to the extracellular soluble target protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein, wherein the membrane-associated protein is associated with a target cell; and wherein the contacting of the extracellular soluble target protein and the membrane- associated protein with the binding agent leads to degradation of the soluble target protein.

2. The method of claim 1, wherein the binding agent comprises a bispecific, or a multispecific binding agent.

3. The method of claim 1 or 2, wherein the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, an immunocytokine, an IgG-cytokine, a knob and hole bispecific IgG, a Fc-Fab, a Fc-cytokine, a scFv-Fc-cytokine, a Fab-cytokine, or a knob and hole bispecific Fc-Fab.

4. The method of any one of claims 1-3, wherein the binding agent comprises a third binding domain.

5. The method of any one of claims 1-4, wherein the second binding domain comprises a second binding domain variable heavy chain and a second binding domain variable light chain.

6. The method of any one of claims 1-4, wherein the second binding domain comprises a cytokine, or a biologically active fragment thereof, selected from the group comprising CXCL12, CCL1, CCL2, CCL3, CCL3L1, CCL4, CC4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL11, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, vMIPII, and vCXCl.

7. The method of any one of claims 1-6, wherein the second binding domain specifically binds to one membrane-associated protein.

8. The method of any one of claims 1-7, wherein the membrane-associated protein comprises single-pass and multi-pass membrane proteins.

9. The method of any one of claims 1-8, wherein the membrane-associated protein comprises a tissue-type specific protein.

10. The method of any one of claims 1-9, wherein the membrane-associated protein comprises a cell-type specific protein.

11. The method of any one of claims 1-10, wherein the membrane-associated protein comprises an endogenous internalizing receptor or a membrane-associated ubiquitin E3 ligase.

12. The method of claim 11, wherein the endogenous internalizing receptor comprises targeting receptors or recycling receptors.

13. The method of any one of claims 1-12, wherein the membrane-associated protein is selected from one or more of the following: HER2, CD30, CXCR7, EPHA2, CCR8, 0X40, CD79B, Nectin-4, BCMA, integrin 07, IL1RAP, EGFR, CD33, MUC1, ITGB6, CEACAM5, CDH17, CD20, CD22, CD19, BAFF-R, CD38, TROP2, B7-H3, MARCH- 1, MARCH-8, MARCH-9, GRAIL, Tissue factor, FOLR1, CD45, c-Kit, and TFRC.

14. The method of any one of claims 1-13, wherein the target cell comprises a microglia cell, endothelial cell, smooth muscle cell, immune cell, plasma cell, B cell, neuronal cell, innate immune cell, lymphocyte, monocyte, granulocyte, T cell, regulatory T cell, effector T cell, activated CD8 T cell, CD8+ T cell, CD4+ T cell, Th 17 cell, macrophage, or dendritic cell.

15. The method of any one of claims 1-14, wherein the membrane-associated protein is enriched on a regulatory T cell when compared to an effector T cell.

16. The method of any one of claims 1-15, wherein the membrane-associated protein is enriched on an effector T cell when compared to a regulatory T cell.

17. The method of any one of claims 1-16, wherein the first binding domain binds to an epitope of the soluble target protein.

18. The method of any one of claims 1-17, wherein the soluble target protein comprises an aggregated protein.

19. The method of any one of claims 1-18, wherein the soluble target protein is selected from one or more of the following: an immune checkpoint protein, an immunomodulatory protein, an inflammatory cytokine, an autoantibody, a shed receptor, and a neuronal aggregate protein.

20. The method of any one of claims 1-19, wherein the soluble target protein is selected from one or more of the following: BAFF, IgG4, TNFa, MICA / B, IL-23, IL-1, IL-2, IL-4, IL-6, IL- 12, IFNy, and A0 protein.

21. The method of claim 20, wherein the soluble target protein comprises BAFF.

22. The method of claim 20, wherein the soluble target protein comprises IgG423. The method of claim 20, wherein the soluble target protein comprises TNFa.

24. The method of claim 20, wherein the soluble target protein comprises MICA / B.

25. The method of claim 20, wherein the soluble target protein comprises IL-23.

26. The method of claim 25, wherein the membrane-associated protein comprises integrin P7.

27. The method of claim 25 or 26, wherein the target cell comprises a T cell.

28. The method of claim 20, wherein the soluble target protein comprises IL-1.

29. The method of claim 20, wherein the soluble target protein comprises IL-2.

30. The method of claim 20, wherein the soluble target protein comprises IL-4.

31. The method of claim 20, wherein the soluble target protein comprises IL-6.

32. The method of claim 20, wherein the soluble target protein comprises IL-12.

33. The method of claim 20, wherein the soluble target protein comprises IFNy.

34. The method of claim 20, wherein the soluble target protein comprises A0 protein.

35. The method of claim 34, wherein the membrane-associated protein is not an Fey receptor.

36. The method of claim 34 or 35, wherein the A0 protein is an intracellular or extracellular A0 protein.

37. The method of any one of claims 1-36, wherein contacting of the second binding domain to the membrane-associated protein results in the internalization of the membrane-associated protein and the binding agent.

38. The method of any one of claims 1-37, wherein contacting of the first binding domain to the soluble target protein, and the subsequent or simultaneous contacting of the second binding domain to the membrane-associated protein, results in the internalization of the membrane-associated protein, the binding agent, and the soluble target protein, and wherein the soluble target protein is then degraded.

39. The method of any one of claims 1-38, wherein the membrane-associated protein is recycled to the target cell surface following the internalization of the binding agent.

40. A method of degrading a membrane-bound target protein, the method comprising: contacting the membrane-bound target protein and a membrane-associated protein with a binding agent, wherein the binding agent comprises:(i) a first binding domain that specifically binds to the membrane-bound target protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein, wherein the membrane-associated protein is associated with a target cell; and wherein the contacting of the membrane-bound target protein and the membrane- associated with the binding agent leads to degradation of the membrane-bound target protein.

41. The method of claim 40, wherein the binding agent comprises a bispecific, or a multispecific binding agent.

42. The method of claim 40 or 41, wherein the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, an immunocytokine, an IgG-cytokine, a knob and hole bispecific IgG, a Fc-Fab, a Fc-cytokine, a scFv-Fc-cytokine, a Fab-cytokine, or a knob and hole bispecific Fc-Fab.

43. The method of any one of claims 40-42, wherein the second binding domain comprises a second binding domain variable heavy chain and a second binding domain variable light chain.

44. The method of any one of claims 40-42, wherein the second binding domain comprises a cytokine, or a biologically active fragment thereof, selected from the group comprising CXCL12, CCL1, CCL2, CCL3, CCL3L1, CCL4, CC4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL11, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, vMIPII, and vCXCl.

45. The method of any one of claims 40-44, wherein the second binding domain specifically binds to one membrane-associated protein.

46. The method of any one of claims 40-45, wherein the membrane-associated protein comprises single-pass and multi-pass membrane proteins.

47. The method of any one of claims 40-46, wherein the membrane-associated protein comprises a tissue-type specific protein.

48. The method of any one of claims 40-47, wherein the membrane-associated protein comprises a cell-type specific protein.

49. The method of any one of claims 40-48, wherein the membrane-associated protein comprises an endogenous internalizing receptor or a membrane-associated ubiquitin E3 ligase.

50. The method of claim 49, wherein the endogenous internalizing receptor comprises targeting receptors or recycling receptors.

51. The method of any one of claims 40-50, wherein the membrane-associated protein is selected from one or more of the following: HER2, CD30, CXCR7, EPHA2, CCR8, 0X40, CD79B, Nectin-4, BCMA, integrin 07, IL1RAP, EGFR, CD33, MUC1, ITGB6, CEACAM5, CDH17, CD20, CD22, CD19, BAFF-R, CD38, TROP2, B7-H3, MARCH- 1, MARCH-8, MARCH-9, GRAIL, Tissue factor, FOLR1, CD45, c-Kit, and TFRC.

52. The method of any one of claims 40-51, wherein the target cell comprises a microglia cell, endothelial cell, smooth muscle cell, immune cell, plasma cell, B cell, neuronal cell, innate immune cell, lymphocyte, monocyte, granulocyte, regulatory T cell, T cell, effector T cell, activated CD8 T cell, CD8+ T cell, CD4+ T cell, Th 17 cell, macrophage, or dendritic cell.

53. The method of any one of claims 40-52, wherein the membrane-associated protein is enriched on a regulatory T cell when compared to an effector T cell.

54. The method of any one of claims 40-53, wherein the membrane-associated protein is enriched on an effector T cell when compared to a regulatory T cell.

55. The method of any one of claims 40-54, wherein the first binding domain binds to an epitope of the membrane-bound target protein.

56. The method of any one of claims 40-55, wherein the membrane-bound target protein is selected from one or more of the following: an immune checkpoint protein, an immunomodulatory protein, and an autoantibody.

57. The method of any one of claims 40-56, wherein the membrane-bound target protein comprises a tissue-type specific protein.

58. The method of any one of claims 40-57, wherein the membrane-bound target protein comprises a cell-type specific protein.

59. The method of any one of claims 40-58, wherein the membrane-bound target protein comprises multiple ligand-binding sites.

60. The method of any one of claims 40-59, wherein the membrane-bound target protein is selected from one or more of the following: MerTK, PD-1, ICOS, TIM3, B7H4, RAGE, HLA DQ2.5, GSDMD, TLR7, CD86, MHC-II, MHC-I, ICAM, c-Kit, CD83, CD3(^ and a4 integrin protein.

61. The method of claim 60, wherein the membrane-bound target protein comprises MerTK.

62. The method of claim 60, wherein the membrane-bound target protein comprises PD- 1.

63. The method of claim 62, wherein the membrane-associated protein comprises CD30.

64. The method of claim 60, wherein the membrane-associated protein comprises CCR8.

65. The method of claim 60, wherein the membrane-associated protein comprises 0X40.

66. The method of any one of claims 62-65, wherein the target cell comprises a regulatory T cell.

67. The method of claim 60, wherein the membrane-bound target protein comprises ICOS.

68. The method of claim 67, wherein the membrane-associated protein comprises IL I R.AP.

69. The method of claim 67 or 68, wherein the target cell comprises an effector T cell.

70. The method of claim 60, wherein the membrane-bound target protein comprises TIM3.

71. The method of claim 60, wherein the membrane-bound target protein comprises B7H4.

72. The method of claim 60, wherein the membrane-bound target protein comprises RAGE.

73. The method of claim 60, wherein the membrane-bound target protein comprises HLA DQ2.5.

74. The method of claim 60, wherein the membrane-bound target protein comprises GSDMD.

75. The method of claim 60, wherein the membrane-bound target protein comprises TLR7.

76. The method of claim 60, wherein the membrane-bound target protein comprises MHC-II.

77. The method of claim 60, wherein the membrane-bound target protein comprises CD86.

78. The method of claim 76 or 77, wherein the membrane-associated protein comprises MARCH-8.

79. The method of claim 77, wherein the membrane-associated protein comprises MARCH- 1.

80. The method of any one of claims 76-79, wherein the target cell comprises a T cell.

81. The method of claim 60, wherein the membrane-bound target protein comprises MHC-I.

82. The method of claim 60, wherein the membrane-bound target protein comprises ICAM.

83. The method of claim 81 or 82, wherein the membrane-associated protein comprises MARCH-9.

84. The method of any one of claims 81-83, wherein the target cell comprises a T cell.

85. The method of claim 60, wherein the membrane-bound target protein comprises CD83.

86. The method of claim 60, wherein the membrane-bound target protein comprises CD387. The method of claim 85 or 86, wherein the membrane-associated protein comprises GRAIL.

88. The method of any one of claims 85-87, wherein the target cell comprises a T cell.

89. The method of claim 60, wherein the membrane-bound target protein comprises a4 integrin.

90. The method of any one of claims 40-89, wherein the membrane-bound target protein is located on the target cell.

91. The method of any one of claims 40-90, wherein the membrane-bound target protein is not located on the surface of a target cell.

92. The method of any of claims 40-91, wherein the membrane-bound target protein is located on an organelle within the target cell.

93. The method of any of claims 40-92, wherein the membrane-bound target protein is a lysosomal membrane-associated protein.

94. The method of any one of claims 40-94, wherein contacting of the second binding domain to the membrane-associated protein results in the internalization of the membrane-associated protein and the binding agent.

95. The method of any one of claims 40-94, wherein contacting of the first binding domain to the membrane-bound target protein, and the subsequent or simultaneous contacting of the second binding domain to the membrane-associated protein, results in the internalization of the membrane-associated protein, the binding agent, and the membrane-bound target protein, and wherein the membrane-bound target protein is then degraded.

96. The method of any one of claims 40-95, wherein the membrane-associated protein is recycled to the target cell surface following the internalization of the binding agent.

97. A method of degrading a BAFF protein, the method comprising: contacting the BAFF protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the BAFF protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the BAFF protein is internalized with the membrane-associated protein into a target cell and the BAFF protein is degraded.

98. A method of degrading an IgG4 protein, the method comprising: contacting the IgG4 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the IgG4 protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the IgG4 protein is internalized with the membrane-associated protein into a target cell and the IgG4 protein is degraded.

99. A method of degrading a TNFa protein, the method comprising: contacting the TNFa protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the TNFa protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein;wherein the TNFa protein is internalized with the membrane-associated protein into a target cell and the TNFa protein is degraded.

100. A method of degrading a MICA / B protein, the method comprising: contacting the MICA / B protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the MICA / B protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the MICA / B protein is internalized with the membrane-associated protein into a target cell and the MICA / B protein is degraded.

101. A method of degrading an IL-23 protein, the method comprising: contacting the IL-23 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the IL-23 protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the IL-23 protein is internalized with the membrane-associated protein into a target cell and the IL-23 protein is degraded.

102. A method of degrading an IL-1 protein, the method comprising: contacting the IL-1 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the IL-1 protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the IL-1 protein is internalized with the membrane-associated protein into a target cell and the IL-1 protein is degraded.

103. A method of degrading an IL-6 protein, the method comprising: contacting the IL-6 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the IL-6 protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the IL-6 protein is internalized with the membrane-associated protein into a target cell and the IL-6 protein is degraded.

104. A method of degrading a IFNg protein, the method comprising: contacting the IFNg protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the IFNg protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the IFNg protein is internalized with the membrane-associated protein into a target cell and the IFNg protein is degraded.

105. A method of degrading an A0 protein, the method comprising: contacting the A0 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the A0 protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the A0 protein is internalized with the membrane-associated protein into a target cell and the A0 protein is degraded.

106. The method of claim 103, wherein the membrane-associated protein is not an Fey receptor.

107. The method of claim 103 or 104, wherein the A0 protein is an intracellular or extracellular A0 protein.

108. A method of degrading a MerTK protein, the method comprising: contacting the MerTK protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the MerTK protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the MerTK protein is internalized with the membrane-associated protein into a target cell and the MerTK protein is degraded.

109. A method of degrading a PD-1 protein, the method comprising: contacting the PD-1 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the PD-1 protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the PD-1 protein is internalized with the membrane-associated protein into a target cell and the PD-1 protein is degraded.

110. A method of degrading a ICOS protein, the method comprising: contacting the ICOS protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the ICOS protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the ICOS protein is internalized with the membrane-associated protein into a target cell and the ICOS protein is degraded.

111. A method of degrading a TIM3 protein, the method comprising: contacting the TIM3 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the TIM3 protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the TIM3 protein is internalized with the membrane-associated protein into a target cell and the TIM3 protein is degraded.

112. A method of degrading a B7H4 protein, the method comprising:contacting the B7H4 protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the B7H4 protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the B7H4 protein is internalized with the membrane-associated protein into a target cell and the B7H4 protein is degraded.

113. A method of degrading a RAGE protein, the method comprising: contacting the RAGE protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the RAGE protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the RAGE protein is internalized with the membrane-associated protein into a target cell and the RAGE protein is degraded.

114. A method of degrading an a4 integrin protein, the method comprising: contacting the a4 integrin protein and a membrane-associated protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the a4 integrin protein, and(ii) a second binding domain that specifically binds to the membrane- associated protein; wherein the a4 integrin protein is internalized with the membrane-associated protein into a target cell and the a4 integrin protein is degraded.

115. The method of any one of claims 95-112, wherein the membrane-associated protein comprises single-pass and multi-pass membrane proteins.

116. The method of any one of claims 95-113, wherein the membrane-associated protein comprises a tissue-type specific protein.

117. The method of any one of claims 95-114, wherein the membrane-associated protein comprises a cell-type specific protein.

118. The method of any one of claims 95-115, wherein the membrane-associated protein comprises an endogenous internalizing receptor or a membrane-associated ubiquitin E3 ligase.

119. The method of claim 116, wherein the endogenous internalizing receptor comprises targeting receptors and recycling receptors.

120. The method of any one of claims 95-117, wherein the membrane-associated protein is selected from one or more of the following: HER2, CD30, CXCR7, EPHA2, CCR8, 0X40, CD79B, Nectin-4, BCMA, integrin 07, IL1RAP, EGFR, CD33, MUC1, ITGB6, CEACAM5, CDH17, CD20, CD22, CD19, BAFF-R, CD38, TROP2, B7-H3, MARCH- 1, MARCH-8, MARCH-9, GRAIL, Tissue factor, FOLR1, CD45, c-Kit, and TFRC.

121. The method of claim 118, wherein the membrane-associated protein comprises IL I RAP.

122. The method of claim 118, wherein the membrane-associated protein comprises integrin 07.

123. A method of degrading an IL-23 protein, the method comprising: contacting the IL-23 protein and an integrin 07 protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the IL-23 protein, and(ii) a second binding domain that specifically binds to the integrin 07 protein; wherein the IL-23 protein is internalized with the integrin 07 protein into a target cell and the IL-23 protein is degraded.

124. A method of degrading an ICOS protein, the method comprising: contacting the ICOS protein and an ILIRAP protein with a binding agent; and wherein the binding agent comprises:(i) a first binding domain that specifically binds to the ICOS protein, and(ii) a second binding domain that specifically binds to the ILIRAP protein; wherein the ICOS protein is internalized with the ILIRAP protein into a target cell and the ICOS protein is degraded.

125. The method of any one of claims 95-122, wherein the binding agent comprises a bispecific, or a multi-specific binding agent.

126. The method of any one of claims 95-123, wherein the binding agent is a multispecific antibody, a bispecific diabody, a bispecific Fab2, bispecific camelid antibody, a bispecific peptibody scFv-Fc, a bispecific IgG, an immunocytokine, an IgG-cytokine, a knob and hole bispecific IgG, a Fc-Fab, a Fc-cytokine, a scFv-Fc- cytokine, a Fab-cytokine, or a knob and hole bispecific Fc-Fab.

127. The method of any one of claims 95-124, wherein the second binding domain comprises a second binding domain variable heavy chain and a second binding domain variable light chain.

128. The method of any one of claims 95-124, wherein the second binding domain comprises a cytokine, or a biologically active fragment thereof, selected from the group comprising CXCL12, CCL1, CCL2, CCL3, CCL3L1, CCL4, CC4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL11, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, vMIPII, and vCXCl.

129. The method of any one of claims 95-126, wherein the second binding domain specifically binds to one membrane-associated protein.

130. The method of any one of claims 95-127, wherein the target cell comprises a microglia cell, endothelial cell, smooth muscle cell, immune cell, plasma cell, B cell, neuronal cell, innate immune cell, lymphocyte, monocyte, granulocyte, regulatory T cell, T cell, effector T cell, activated CD8 T cell, CD8+ T cell, CD4+ T cell, Th 17 cell, macrophage, or dendritic cell.

131. The method of any one of claims 95-128, wherein contacting of the second binding domain to the membrane-associated protein results in the internalization of the membrane-associated protein and the binding agent.

132. The method of any one of claims 95-129, wherein the membrane-associated protein is recycled to the target cell surface following the internalization of the binding agent.

133. A pharmaceutical composition, comprising the binding agent of any one of claims 1- 130, and a pharmaceutically acceptable excipient.

134. A method of treating a disorder in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective amount of the pharmaceutical composition of claim 131.

135. A method of treating a disorder in a subject, wherein the method comprises depleting a target protein in the subject by administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 131.

136. The method of claim 133, wherein the target protein is selected from one or more of the following: BAFF, IgG4, TNFa, MICA / B, IL-23, IL-1, IL-2, IL-4, IL-6, IL-12, IFNg, Ap protein, MerTK, PD-1, ICOS, TIM3, B7H4, RAGE, HLA DQ2.5, GSDMD, TLR7and a4 integrin protein.

137. The method of claim 134, wherein the target protein comprises BAFF.

138. The method of claim 134, wherein the target protein comprises IgG4139. The method of claim 134, wherein the target protein comprises TNFa.

140. The method of claim 134, wherein the target protein comprises MICA / B.

141. The method of claim 134, wherein the target protein comprises IL-23.

142. The method of claim 134, wherein the target protein comprises IL-1.

143. The method of claim 134, wherein the target protein comprises IL-2.

144. The method of claim 134, wherein the target protein comprises IL-4.

145. The method of claim 134, wherein the target protein comprises IL-6.

146. The method of claim 134, wherein the target protein comprises IL-12.

147. The method of claim 134, wherein the target protein comprises IFNg.

148. The method of claim 134, wherein the target protein comprises A0 protein.

149. The method of claim 134, wherein the target protein comprises MerTK.

150. The method of claim 134, wherein the target protein comprises PD-1.

151. The method of claim 134, wherein the target protein comprises ICOS.

152. The method of claim 134, wherein the target protein comprises TIM3.

153. The method of claim 134, wherein the target protein comprises B7H4.

154. The method of claim 134, wherein the target protein comprises RAGE.

155. The method of claim 134, wherein the target protein comprises HLA DQ2.5.

156. The method of claim 134, wherein the target protein comprises GSDMD.

157. The method of claim 134, wherein the target protein comprises TLR7.

158. The method of claim 134, wherein the target protein comprises a4 integrin.

159. The method of any one of claims 132-156, wherein the subject is a mammal.

160. The method of any one of claims 132-157, wherein the subject is a human.

161. The method of any one of claims 132-158, wherein the disorder comprises a neoplastic disorder, an inflammatory-related disease, an autoimmune disease, a cardiovascular disease, or a neurological disorder.

162. The method of claim 159, wherein the neoplastic disorder comprises breast cancer, B cell lymphoma, pancreatic cancer, Hodgkin’s lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin’s B-cell (B-NHL), melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, or colorectal cancer.

163. The method of claim 159, wherein the inflammatory-related disease comprises inflammatory intestinal disease, type 1 diabetes, arthritis, rheumatoid arthritis, psoriasis, lupus, Crohn's disease, colitis, or autoantibody-driven pathologies.

164. The method of claim 159, wherein the autoimmune disease comprises SLE, scleroderma, addison disease, celiac disease - sprue, gluten-sensitive enteropathy, dermatomyositis, Graves disease, hashimoto thyroiditis, type 1 diabetes, lupus, alopecia areata, pemphigus vulgaris, inflammatory bowel disease, crohn disease, ulcerative colitis, multiple sclerosis, or myasthenia gravis.

165. The method of claim 159, wherein the neurological disorder comprises Parkinson's disease, Alzheimer's disease, a neurodegeneration disease, or multiple sclerosis.

166. A method of depleting BAFF in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

167. A method of depleting IgG4 in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

168. A method of depleting TNFa in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

169. A method of depleting MICA / B in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

170. A method of depleting IL-23 in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

171. A method of depleting IL-1 in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

172. A method of depleting IL-6 in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

173. A method of depleting IFNg in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

174. A method of depleting A0 protein in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

175. A method of depleting MerTK in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

176. A method of depleting PD-1 expressing regulatory T cells in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

177. A method of depleting ICOS expressing T cells in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

178. A method of depleting TIM3 in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

179. A method of depleting B7H4 in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

180. A method of depleting RAGE expressing innate immune cells in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

181. A method of depleting a4 integrin in a human patient comprising administering to a human in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 131.

182. The method of any one of claims 40-59, wherein the membrane-bound target protein is an intracellular protein.

183. The method of any one of claims 1-19, wherein the soluble target protein is an intracellular protein.

184. The method of claim 182 or 183, wherein the method further comprises modulating an intracellular protein comprising targeting an upstream cell surface receptor with a bispecific or a multispecific antibody.

185. The method of any one of claims 182-184, wherein the intracellular protein is a cell signaling protein.

186. The method of claim 185, wherein the cell signaling protein is selected from FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lek, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3 / 6, MKK4 / 7, ASKl,Cot, NIK, Bub, Myt 1, Weel, Casein kinases, PDK1, SGK1, SGK2, SGK3, Aktl, Akt2, Akt3, p90Rsks, p70S6 Kinase, Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs, Chkl, Chk2, LKB-1, MAPKAPKs, Piml, Pim2, Pim3, IKKs, Cdks, Inks, Erks, IKKs, GSK3a, GSK3P, Cdks, CLKs, PKR, PI3 -Kinase, mTor, SAPK / JNK1,2,3, p38s, PKR, DNA-PK, ATM, or ATR.

187. The method of claim 185 or 186, wherein the cell signaling receptor is selected from H-Ras, K-Ras, orN-Ras.

188. The method of any one of claims 184-187, wherein the modulating of the intracellular protein comprises downregulating, upregulating, or post-translationally modifying the intracellular protein.

189. The method of any one of claims 184-188, wherein the modulating of the intracellular protein comprises modulating the scaffolding of the intracellular protein.

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