Tunable darpin-based and / or Anti-angiogenic and / or immunomodulatory nanoparticle conjugates, cellular immunoherapeutics, and uses thereof

Abstract

Disclosed herein are nanoparticle conjugates (e.g., anti-angiogenic and / or immunomodulatory nanoparticle conjugates, e.g., high-affinity Designed Ankyrin Repeat Protein (DARPin)-based binders targeting one or more cancer and / or immune receptors). The nanoparticle conjugates are useful for therapeutics and / or diagnostics and have a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution).

Description

Attorney Docket No. 2018488-0014 TUNABLE DARPin-BASED AND / OR ANTI-ANGIOGENIC AND / OR IMMUNOMODULATORY NANOPARTICLE CONJUGATES, CELLULAR IMMUNOHERAPEUTICS, AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application number 63 / 779,380, filed March 28, 2025, and U.S. Provisional Application number 63 / 653,349, filed May 30, 2024, the disclosures of which are incorporated by reference herein in their entireties. FIELD OF INVENTION

[0002] This invention relates generally to nanoparticle conjugate compositions for the treatment of disease or conditions in subjects. More specifically, in certain embodiments, the invention relates to therapies for treating cancer. BACKGROUND

[0003] Brain metastases are the most common type of intracranial tumor, with an estimated incidence of 70,000-400,000 cases per annum. Up to 30% of patients with advanced breast cancer (BC) develop brain metastases. The incidence of breast cancer brain metastases (BCBM) is rising, in part, due to a lack of effective treatments, improved control of extracranial disease by systemic therapies, poor drug penetrability of the blood-brain barrier (BBB), and poorly understood differences in the microenvironments of brain metastases relative to early-stage disease. Surgical intervention stereotactic radiosurgery (SRS), chemotherapy, and targeted therapies have lacked efficacy, resulting in a reduced quality of life and median survival times of less than one year.

[0004] Moreover, corticosteroids, and their synthetic analogs, e.g., dexamethasone (DEX), are routinely used in the clinic to decrease edema and mitigate inflammatory side effects associated with SRS and symptomatic metastases. However, they promote a host of toxic and debilitating neurological symptoms, including headache, confusion, immune suppression and hyperglycemia. Paradoxically, these agents also reduce survival in malignant brain tumors and exert deleterious effects on the formation and progression of metastases– a consequence of activating corticosteroid-mediated pathways and receptors at distant metastatic sites. These data highlight concerns regarding the - 1 - 12799878v1Attorney Docket No. 2018488-0014 utilization of exogenous corticosteroids in cancer patients and the importance of developing non- steroidal agents that can successfully reduce neuroinflammation and edema.

[0005] Accordingly, there is a need for a therapeutic strategy to improve tumor control and counteract adaptive resistance mechanisms, and / or reduce deaths in subjects with cancer (e.g., BCBM). SUMMARY OF THE INVENTION

[0006] The present disclosure describes nanoparticle conjugates for the treatment of diseases and conditions. For example, the present disclosure describes Designed Ankyrin Repeat Protein (DARPin)-based nanoparticle conjugates. The present disclosure also describes anti-angiogenic and / or immunomodulatory nanoparticle conjugates (“AAINCs”).

[0007] The described (DARPin)-based nanoparticle conjugates can target (e.g., simultaneously) tumor cell surface receptors and soluble growth factors to enhance treatment specificity and overcome resistance in cancer therapy. The described DARPin-based nanoparticle conjugates can be used alone or in combination with other therapeutic modalities as described herein. Importantly, site-specific conjugation of DARPins to a nanoparticle had minimal impact on antigen recognition, underscoring the structural resilience of the binding domains. Thus, the present disclosure provides nanoparticle conjugates having a flexible and scalable DARPin scaffold (e.g., bispecific DARPin scaffold) that supports efficient, plug-and-play conjugation of imaging and therapeutic payloads without compromising function. Accordingly, the preserved dual-targeting capability across a wide range of modifications positions this platform as a promising foundation for precision oncology tools, including image-guided diagnostics, targeted radiotherapy, and site-specific drug delivery.

[0008] The described nanoparticle conjugates can also be used alone or in combination with other therapeutic modalities (e.g., low-dose radiation therapy), for example, to suppress neuroinflammation and / or edema. For instance, the described AAINCs provide a versatile platform that can be used to target an angiogenic protein (e.g., an angiogenic protein involved in angiogenesis).

[0009] The disclosed nanoparticle conjugates can also be used to mitigate the onset and / or progression of inflammatory changes (e.g., edema) that arise with radiation necrosis, generated by using high-dose radiation therapy. The present disclosure also provides for quantification of edema volumes, tumor size, and other magnetic resonance imaging (MRI) functional parameters using - 2 - 12799878v1Attorney Docket No. 2018488-0014 multiparametric MRI. Importantly, the described nanoparticle conjugates can be used as precision oncology tools, including image-guided diagnostics, targeted radiotherapy, and site-specific drug delivery.

[0010] In one aspect, the invention is directed to a Designed Ankyrin Repeat Protein (DARPin) nanoparticle conjugate comprising: a nanoparticle; and one or more DARPins conjugated to the nanoparticle (e.g., wherein the one or more DARPins prior to conjugation each comprises a moiety (e.g., an azide moiety) for conjugation to the nanoparticle), wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution).

[0011] In certain embodiments, the one or more DARPins comprises one or more DARPin binders (e.g., a monospecific DARPin, e.g., bispecific DARPin, e.g., a trispecific DARPin). In certain embodiments, the one or more DARPin binders comprises a HER2-binding domain (e.g., that selectively binds to HER2-over expressing tumor cells). In certain embodiments, the one or more DARPin binders comprises a CD3-binding domain (e.g., that binds to a CD3e chain on a T-cell receptor (TCR) complex of T cells). In certain embodiments, the one or more DARPin binders comprises an EGFR-binding domain. In certain embodiments, the one or more DARPin binders comprises an a4-1BB-binding domain. In certain embodiments, the one or more DARPin binders comprises an EpCam-binding domain. In certain embodiments, the one or more DARPin binders comprises a VEGF binding domain (e.g., for inhibition of tumor angiogenesis). In certain embodiments, the one or more DARPin binders comprises a HER2-binding domain and a CD3-binding domain. In certain embodiments, the one or more DARPin binders comprises a HER2-binding domain and a VEGF-binding domain. In certain embodiments, the one or more DARPin binders comprises a HER2-binding domain, an EGFR-binding domain, and a VEGF-binding domain. In certain embodiments, the one or more the one or more DARPin binders comprises a CD3-binding domain, an EGFR-binding domain, and a HER2-binding domain. In certain embodiments, the nanoparticle conjugate penetrates the blood brain barrier (BBB). In certain embodiments, the one or more DARPins is covalently or non-covalently bonded to the nanoparticle via a linker or covalently or non-covalently bonded directly to the nanoparticle, or associated with the nanoparticle or a moiety surrounding the nanoparticle, e.g., via van der Waals forces. - 3 - 12799878v1Attorney Docket No. 2018488-0014

[0012] In another aspect, the invention is directed to an anti-angiogenic and / or immunomodulatory nanoparticle conjugate (“AAINC”) comprising: a nanoparticle; and one or more antibody fragments (e.g., fragments of a single member or fragments of different members) conjugated to the nanoparticle (e.g., wherein the one or more antibody fragments prior to conjugation each comprises a moiety (e.g., a cysteine moiety) for conjugation to the nanoparticle), wherein the one or more antibody fragments targets an angiogenic protein, wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers.

[0013] In certain embodiments, the angiogenic protein comprises a VEGF protein (e.g., a VEGF-A protein, a VEGF-B protein, a VEGF-C protein, a VEGF-D protein, a VEGF-E protein, or a VEGF-F protein, or any combination thereof). In certain embodiments, the one or more antibody fragments comprises anti-VEGF (e.g., anti-VEGF-A, anti-VEGF-B, or a combination thereof) (e.g., anti-VEGF-A-scFv, anti-VEGF-B-scFv, anti-VEGF-C-scFv, anti-VEGF-D-scFv, anti-VEGF-E-scFv, or anti-VEGF-F-scFv, or a combination thereof). In certain embodiments, the one or more antibody fragments is a member selected from the set consisting of a recombinant antibody fragment (Fabs), a single chain variable fragment (scFv), and a single domain antibody (sdAb) fragment. In certain embodiments, the one or more antibody fragments is a single chain variable fragment (scFv). In certain embodiments, the one or more antibody fragments is covalently or non-covalently bonded to the nanoparticle via a linker or covalently or non-covalently bonded directly to the nanoparticle, or associated with the nanoparticle or a moiety surrounding the nanoparticle, e.g., via van der Waals forces.

[0014] In certain embodiments, the one or more antibody fragments comprises from 1 to 20 antibody fragments (e.g., total of a single member or total of different members) (e.g., 1 to 15 antibody fragments, e.g., 1 to 10 antibody fragments, e.g., 2 to 15 antibody fragments, e.g., 5 to 10 antibody fragments, e.g., about 6 to 8 antibody fragments, e.g., about 1 to 5 antibody fragments, e.g., about 1 to 2 antibody fragments) conjugated to the nanoparticle. In certain embodiments, the one or more antibody fragments comprises a molecular range no greater than 50 kDa (e.g., no greater than 40 kDa, e.g., no greater than 30 kDa, e.g., no greater than 25 kDa).

[0015] In certain embodiments, the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm, e.g., from 4 to 8 nm. - 4 - 12799878v1Attorney Docket No. 2018488-0014 (e.g., wherein the nanoparticle conjugate has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm).

[0016] In certain embodiments, the nanoparticle is coated with an organic polymer (e.g., polyethylene glycol (PEG)) (e.g., a partial or complete coating). In certain embodiments, the nanoparticle conjugate comprises a radiolabel (e.g.,89Zr) (e.g., an imaging and / or a therapeutic radioisotope). In certain embodiments, the nanoparticle conjugate comprises a first chelator (e.g., deferoxamine (DFO)). In certain embodiments, the nanoparticle comprises silica. In certain embodiments, the nanoparticle comprises a silica core. In certain embodiments, the nanoparticle comprises a silica-based core and a silica shell surrounding at least a portion of the core. In certain embodiments, the nanoparticle comprises a fluorescent compound within the core.

[0017] In certain embodiments, the nanoparticle comprises a silica composition such that ferroptosis is not induced (e.g., ferroptosis is switched “off”). In certain embodiments, the nanoparticles have a silica composition such that ferroptosis, other cell death processes (e.g., pyroptosis, autophagy), and apoptosis may be induced (e.g., ferroptosis is not switched “off”).

[0018] In certain embodiments, the nanoparticle conjugate, further comprises a therapeutic agent. In certain embodiments, the therapeutic agent is associated (e.g., covalently, e.g., non- covalently) to the nanoparticle. In certain embodiments, the therapeutic agent is associated (e.g., covalently, e.g., non-covalently) with the one or more DARPins or with the one or more DARPins and the nanoparticle. In certain embodiments, the therapeutic agent is associated (e.g., covalently, e.g., non-covalently) with the one or more antibody fragments or with the one or more antibody fragments and the nanoparticle. In certain embodiments, the nanoparticle further comprises a targeting ligand.

[0019] In another aspect, the invention is directed to a method of treating a disease or condition and / or mitigating the onset and / or progression of a disease (e.g., inflammation, e.g., neuroinflammation, e.g., edema, e.g., radiation necrosis, e.g., cancer, e.g., breast cancer, e.g., brain cancer, e.g., breast cancer brain metastases, e.g., a combination of any one inflammation, neuroinflammation, edema, radiation necrosis, cancer, breast cancer, brain cancer, or breast cancer brain metastases) for a subject (e.g., a human subject) (e.g., a subject diagnosed with and / or suspected - 5 - 12799878v1Attorney Docket No. 2018488-0014 of having and / or having a predisposition for the disease or condition), the method comprising administering to the subject a pharmaceutical composition comprising the nanoparticle conjugate [e.g., to target a particular type of tissue (e.g., brain tissue), e.g., cancer, e.g., breast cancer, e.g., brain cancer, e.g., breast cancer brain metastases]. In certain embodiments, the method comprises administering a therapeutic radioisotope (e.g., wherein the therapeutic radioisotope is attached to a second nanoparticle having a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) (e.g., wherein the radioisotope is attached to the second nanoparticle via a second chelator)) (e.g., wherein the second nanoparticle has a diameter from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm). In certain embodiments, the method comprises administering immunotherapy. In certain embodiments, the immunotherapy comprises administering to a subject a pharmaceutical composition comprising the nanoparticle conjugate. In certain embodiments, the immunotherapy comprises administering an engineered immunotherapy [e.g., for localized treatment, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid primary tumors, e.g., metastatic tumors, e.g., metastatic brain cancer), brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, or other diseases] comprising: an engineered immune cell [e.g., a macrophage, e.g., a neutrophil, e.g., a T cell (e.g., a Steap1 scFv-based CAR-T cell), e.g., a DARPin-based CAR / TCR cells, e.g., an NK cell, e.g., tumor-infiltrating lymphocytes (TILs)]. In certain embodiments, the engineered immune cell comprises (a) a chimeric antigen receptor (CAR) and (b) a bispecific immune cell engager (e.g., a bispecific T-cell engager or “BiTE”, e.g., a bispecific macrophage engager, e.g., a bispecific neutrophil engager). In certain embodiments, the engineered immune cell comprises a CAR T cell comprising one or more targeting ligands (e.g., a MUC16-targeting DARPin). In certain embodiments, the pharmaceutical composition further comprises a carrier. In certain embodiments, the subject has a disease or condition (e.g., inflammation, e.g., neuroinflammation, e.g., edema, e.g., radiation necrosis, e.g., a combination of any one inflammation, neuroinflammation, edema, or radiation necrosis, e.g., cancer, e.g., breast cancer, e.g., brain cancer, e.g., breast cancer brain metastases). In certain embodiments, the subject has received an anti-inflammatory therapy, chemotherapy, radiotherapy, immunotherapy, or engineered cellular therapy (e.g., CAR T cell therapy) (e.g., an engineered immunotherapy [e.g., for localized treatment, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid primary tumors, e.g., metastatic tumors), brain injury (e.g., radiation, - 6 - 12799878v1Attorney Docket No. 2018488-0014 trauma), neurological disorders, inflammatory diseases, or other diseases] comprising: an engineered immune cell [e.g., a macrophage, e.g., a neutrophil, e.g., a T cell (e.g., a Steap1 CAR-T cell), e.g., an NK cell] comprising (a) a chimeric antigen receptor (CAR) and (b) a bispecific immune cell engager (e.g., a bispecific T-cell engager or “BiTE”, e.g., a bispecific macrophage engager, e.g., a bispecific neutrophil engager)) (e.g., wherein the immunotherapeutic and / or engineered cells comprise(s) an engineered immunotherapy [e.g., for localized treatment, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid primary tumors, e.g., metastatic tumors), brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, or other diseases] comprising: an engineered immune cell comprising a CAR T cell comprising one or more targeting ligands, e.g., a MUC16- targeting ligand).

[0020] In another aspect, the invention is directed to a method of treating a disease or condition and / or mitigating the onset and / or progression of a disease (e.g., inflammation, e.g., neuroinflammation, e.g., edema, e.g., radiation necrosis, e.g., cancer, e.g., breast cancer, e.g., brain cancer, e.g., breast cancer brain metastases, e.g., a combination of any one inflammation, neuroinflammation, edema, radiation necrosis, cancer, breast cancer, brain cancer, or breast cancer brain metastases), the method comprising: administering to a subject a first pharmaceutical composition comprising the nanoparticle conjugate, wherein the subject also receives a second pharmaceutical composition. In certain embodiments, the second composition comprises an anti- angiogenic agent, radiotherapeutic agent, a chemotherapeutic agent, an immunotherapeutic agent, or engineered cells (e.g., CAR T cells) (e.g., an engineered immunotherapy [e.g., for localized treatment, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid primary tumors, e.g., metastatic tumors), brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, or other diseases] comprising: an engineered immune cell [e.g., a macrophage, e.g., a neutrophil, e.g., a T cell (e.g., a Steap1 CAR-T cell, e.g., a CAR T cell comprising one or more targeting ligands), e.g., an NK cell] comprising (a) a chimeric antigen receptor (CAR) and (b) a bispecific immune cell engager (e.g., a bispecific T-cell engager or “BiTE”, e.g., a bispecific macrophage engager, e.g., a bispecific neutrophil engager)) (e.g., wherein the immunotherapeutic and / or engineered cells comprise(s) an engineered immunotherapy [e.g., for localized treatment, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid primary tumors, e.g., metastatic tumors), brain injury (e.g., radiation, - 7 - 12799878v1Attorney Docket No. 2018488-0014 trauma), neurological disorders, inflammatory diseases, or other diseases] comprising: an engineered immune cell comprising a CAR T cell comprising one or more targeting ligands, e.g., a MUC16- targeting ligand). In certain embodiments, the first pharmaceutical composition is being used to enhance activity of the second pharmaceutical composition.

[0021] In another aspect, the invention is directed to a method of treating a disease or condition and / or mitigating the onset and / or progression of a disease (e.g., inflammation, e.g., neuroinflammation, e.g., edema, e.g., radiation necrosis, e.g., cancer, e.g., breast cancer, e.g., brain cancer, e.g., breast cancer brain metastases, e.g., a combination of any one inflammation, neuroinflammation, edema, radiation necrosis, cancer, breast cancer, brain cancer, or breast cancer brain metastases), the method comprising: administering to a subject a first pharmaceutical composition comprising the nanoparticle conjugate and a second pharmaceutical composition. In certain embodiments, the second composition comprises an anti-angiogenic agent, a radiotherapeutic agent, a chemotherapeutic agent, an immunotherapeutic agent, or engineered cells (e.g., CAR T cells) (e.g., an engineered immunotherapy [e.g., for localized treatment, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid primary tumors, e.g., metastatic tumors), brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, or other diseases] comprising: an engineered immune cell [e.g., a macrophage, e.g., a neutrophil, e.g., a T cell (e.g., a Steap1 CAR-T cell), e.g., an NK cell] comprising (a) a chimeric antigen receptor (CAR) and (b) a bispecific immune cell engager (e.g., a bispecific T-cell engager or “BiTE”, e.g., a bispecific macrophage engager, e.g., a bispecific neutrophil engager)) (e.g., wherein the immunotherapeutic and / or engineered cells comprise(s) an engineered immunotherapy [e.g., for localized treatment, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid primary tumors, e.g., metastatic tumors), brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, or other diseases] comprising: an engineered immune cell comprising a CAR T cell comprising one or more targeting ligands, e.g., a MUC16- targeting ligand). In certain embodiments, the first pharmaceutical composition is being used to enhance activity of the second pharmaceutical composition.

[0022] In another aspect, the invention is directed to a method of treating a disease or condition and / or mitigating the onset and / or progression of a disease (e.g., inflammation, e.g., neuroinflammation, e.g., edema, e.g., radiation necrosis, e.g., cancer, e.g., breast cancer, e.g., brain - 8 - 12799878v1Attorney Docket No. 2018488-0014 cancer, e.g., breast cancer brain metastases e.g., a combination of any one inflammation, neuroinflammation, edema, radiation necrosis. cancer, breast cancer, brain cancer, or breast cancer brain metastases), the method comprising: administering to a subject a first pharmaceutical composition comprising an anti-angiogenic agent, a radiotherapeutic agent, a chemotherapeutic agent, an immunotherapeutic agent, or engineered cells (e.g., CAR T cells) (e.g., an engineered immunotherapy [e.g., for localized treatment, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid primary tumors, e.g., metastatic tumors), brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, or other diseases] comprising: an engineered immune cell [e.g., a macrophage, e.g., a neutrophil, e.g., a T cell (e.g., a Steap1 CAR-T cell), e.g., an NK cell] comprising (a) a chimeric antigen receptor (CAR) and (b) a bispecific immune cell engager (e.g., a bispecific T-cell engager or “BiTE”, e.g., a bispecific macrophage engager, e.g., a bispecific neutrophil engager)) (e.g., wherein the immunotherapeutic and / or engineered cells comprise(s) an engineered immunotherapy [e.g., for localized treatment, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid primary tumors, e.g., metastatic tumors), brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, or other diseases] comprising: an engineered immune cell comprising a CAR T cell comprising one or more targeting ligands, e.g., a MUC16-targeting ligand), wherein the subject also receives a second pharmaceutical composition comprising the nanoparticle conjugate. In certain embodiments, the second pharmaceutical composition is being used to enhance activity of the first pharmaceutical composition.

[0023] In another aspect, the invention is directed to a method of in vivo imaging (e.g., intraoperative imaging), the method comprising: administering to a subject a composition comprising the nanoparticle conjugate (e.g., such that the nanoparticle conjugate preferentially collects in a particular region, e.g., near or within a particular tissue type, e.g., cancer, e.g., breast cancer, e.g., brain cancer, e.g., breast cancer brain metastases), wherein the nanoparticle conjugate comprises an imaging agent; and detecting (e.g., via PET, X-ray, MRI, CT, etc.) the imaging agent.

[0024] In another aspect, the invention is directed to a method of manufacturing the nanoparticle conjugate, the method comprising: contacting a nanoparticle-Dibenzocyclooctyne (DBCO) with an azide-antibody fragment, thereby producing the nanoparticle conjugate. - 9 - 12799878v1Attorney Docket No. 2018488-0014

[0025] In another aspect, the invention is directed to a composition (e.g., a pharmaceutical composition) comprising one or more agents selected from (1) a nanoparticle conjugate of, (2) a radiotherapeutic agent, (3) a second anti-angiogenic agent, (4) an immunotherapeutic agent, or (5) a combination thereof in a unit dosage effective to treat a disease or condition (e.g., inflammation, e.g., neuroinflammation, e.g., edema, e.g., radiation necrosis, e.g., a combination of any one inflammation, neuroinflammation, edema, or radiation necrosis) in a subject receiving therapy with the agent as part of a combination of (1) to (4) .

[0026] In another aspect, the invention is directed to a Designed Ankyrin Repeat Protein (DARPin) nanoparticle conjugate comprising: a nanoparticle; and one or more DARPins [e.g., wherein the one or more DARPins comprises one or more DARPin binders (e.g., a monospecific DARPin, e.g., bispecific DARPin, e.g., a trispecific DARPin)][e.g., wherein the one or more DARPin binders comprises a HER2-binding domain (e.g., that selectively binds to HER2-over expressing tumor cells)] [e.g., wherein the one or more DARPin binders comprises a CD3-binding domain (e.g., that binds to a CD3e chain on a T-cell receptor (TCR) complex of T cells)] (e.g., wherein the one or more DARPin binders comprises a EGFR-binding domain) (e.g., wherein the one or more DARPin binders comprises an a4-1BB-binding domain) (e.g., wherein the one or more DARPin binders comprises an EpCam- binding domain) [e.g., wherein the one or more DARPin binders comprises a VEGF binding domain (e.g., for inhibition of tumor angiogenesis)] (e.g., wherein the one or more DARPin binders comprises a HER2-binding domain and a CD3-binding domain) (e.g., wherein the one or more DARPin binders comprises a HER2-binding domain and a VEGF-binding domain) (e.g., wherein the one or more DARPin binders comprises a HER2-binding domain, an EGFR-binding domain, and a VEGF-binding domain) (e.g., wherein the one or more DARPin binders comprises a CD3-binding domain, an EGFR- binding domain, and a HER2-binding domain (e.g., wherein the nanoparticle conjugate penetrates the blood brain barrier (BBB)), wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution)(e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm) (e.g., wherein the nanoparticle conjugate has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, - 10 - 12799878v1Attorney Docket No. 2018488-0014 e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) for use in therapy (e.g., inflammation therapy (e.g., neuroinflammation therapy), e.g., edema therapy, e.g., therapy for radiation necrosis, e.g., chemotherapy, e.g., radiotherapy, e.g., cancer, e.g., breast cancer, e.g., brain cancer, e.g., breast cancer brain metastases).

[0027] In another aspect, the invention is directed to a Designed Ankyrin Repeat Protein (DARPin) nanoparticle conjugate comprising: a nanoparticle; and one or more DARPins [e.g., wherein the one or more DARPins comprises one or more DARPin binders (e.g., a monospecific DARPin, e.g., bispecific DARPin, e.g., a trispecific DARPin)][e.g., wherein the one or more DARPin binders comprises a HER2-binding domain (e.g., that selectively binds to HER2-over expressing tumor cells)] [e.g., wherein the one or more DARPin binders comprises a CD3-binding domain (e.g., that binds to a CD3e chain on a T-cell receptor (TCR) complex of T cells)] (e.g., wherein the one or more DARPin binders comprises a EGFR-binding domain) (e.g., wherein the one or more DARPin binders comprises an a4-1BB-binding domain) (e.g., wherein the one or more DARPin binders comprises an EpCam- binding domain) [e.g., wherein the one or more DARPin binders comprises a VEGF binding domain (e.g., for inhibition of tumor angiogenesis)] (e.g., wherein the one or more DARPin binders comprises a HER2-binding domain and a CD3-binding domain) (e.g., wherein the one or more DARPin binders comprises a HER2-binding domain and a VEGF-binding domain) (e.g., wherein the one or more DARPin binders comprises a HER2-binding domain, an EGFR-binding domain, and a VEGF-binding domain) (e.g., wherein the one or more DARPin binders comprises a CD3-binding domain, an EGFR- binding domain, and a HER2-binding domain (e.g., wherein the nanoparticle conjugate penetrates the blood brain barrier (BBB)), wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution)(e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm) (e.g., wherein the nanoparticle conjugate has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) for use in a method of treating a disease or condition and / or mitigating the onset and / or - 11 - 12799878v1Attorney Docket No. 2018488-0014 progression of a disease in a subject, wherein the treating comprises: delivering the nanoparticle conjugate to the subject.

[0028] In another aspect, the invention is directed to a Designed Ankyrin Repeat Protein (DARPin) nanoparticle conjugate comprising: a nanoparticle; and one or more DARPins [e.g., wherein the one or more DARPins comprises one or more DARPin binders (e.g., a monospecific DARPin, e.g., bispecific DARPin, e.g., a trispecific DARPin)][e.g., wherein the one or more DARPin binders comprises a HER2-binding domain (e.g., that selectively binds to HER2-over expressing tumor cells)] [e.g., wherein the one or more DARPin binders comprises a CD3-binding domain (e.g., that binds to a CD3e chain on a T-cell receptor (TCR) complex of T cells)] (e.g., wherein the one or more DARPin binders comprises a EGFR-binding domain) (e.g., wherein the one or more DARPin binders comprises an a4-1BB-binding domain) (e.g., wherein the one or more DARPin binders comprises an EpCam- binding domain) [e.g., wherein the one or more DARPin binders comprises a VEGF binding domain (e.g., for inhibition of tumor angiogenesis)] (e.g., wherein the one or more DARPin binders comprises a HER2-binding domain and a CD3-binding domain) (e.g., wherein the one or more DARPin binders comprises a HER2-binding domain and a VEGF-binding domain) (e.g., wherein the one or more DARPin binders comprises a HER2-binding domain, an EGFR-binding domain, and a VEGF-binding domain) (e.g., wherein the one or more DARPin binders comprises a CD3-binding domain, an EGFR- binding domain, and a HER2-binding domain (e.g., wherein the nanoparticle conjugate penetrates the blood brain barrier (BBB)), wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution)(e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm) (e.g., wherein the nanoparticle conjugate has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) for use in a method of in vivo diagnosis of a disease or condition in a subject, wherein the in vivo diagnosis comprises: delivering the nanoparticle conjugate to the subject; and detecting (e.g., via PET, X-ray, MRI, CT, etc.) the imaging agent. - 12 - 12799878v1Attorney Docket No. 2018488-0014

[0029] In another aspect, the invention is directed to an anti-angiogenic and / or immunomodulatory nanoparticle conjugate (“AAINC”) comprising: a nanoparticle; and one or more antibody fragments (e.g., fragments of a single member or fragments of different members) [e.g., wherein the one or more antibody fragments targets an angiogenic protein (e.g., VEGF, e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, or VEGF-F, or any combination thereof), e.g., wherein the one or more antibody fragments comprises anti-VEGF (e.g., anti-VEGF-A, anti-VEGF-B, or a combination thereof) (e.g., anti-VEGF-A-scFv, anti-VEGF-B-scFv, anti-VEGF-C-scFv, anti-VEGF-D-scFv, anti- VEGF-E-scFv, or anti-VEGF-F-scFv, or a combination thereof)] conjugated to the nanoparticle, wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm) (e.g., wherein the AAINC has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) for use in therapy (e.g., inflammation therapy (e.g., neuroinflammation therapy), e.g., edema therapy, e.g., therapy for radiation necrosis, e.g., chemotherapy, e.g., radiotherapy, e.g., cancer, e.g., breast cancer, e.g., brain cancer, e.g., breast cancer brain metastases).

[0030] In another aspect, the invention is directed to an anti-angiogenic and / or immunomodulatory nanoparticle conjugate (“AAINC”) comprising: a nanoparticle; and one or more antibody fragments (e.g., fragments of a single member or fragments of different members) [e.g., wherein the one or more antibody fragments targets an angiogenic protein (e.g., VEGF, e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, or VEGF-F, or any combination thereof), e.g., wherein the one or more antibody fragments comprises anti-VEGF (e.g., anti-VEGF-A, anti-VEGF-B, or a combination thereof) (e.g., anti-VEGF-A-scFv, anti-VEGF-B-scFv, anti-VEGF-C-scFv, anti-VEGF-D-scFv, anti- VEGF-E-scFv, or anti-VEGF-F-scFv, or a combination thereof)] conjugated to the nanoparticle, wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm) (e.g., wherein the AAINC has an average - 13 - 12799878v1Attorney Docket No. 2018488-0014 diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) for use in a method of treating a disease or condition and / or mitigating the onset and / or progression of a disease in a subject, wherein the treating comprises: delivering the AAINC to the subject.

[0031] In another aspect, the invention is directed to an anti-angiogenic and / or immunomodulatory nanoparticle conjugate (“AAINC”) comprising: a nanoparticle; and one or more antibody fragments (e.g., fragments of a single member or fragments of different members) [e.g., wherein the one or more antibody fragments targets an angiogenic protein (e.g., VEGF, e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, or VEGF-F, or any combination thereof), e.g., wherein the one or more antibody fragments comprises anti-VEGF (e.g., anti-VEGF-A, anti-VEGF-B, or a combination thereof) (e.g., anti-VEGF-A-scFv, anti-VEGF-B-scFv, anti-VEGF-C-scFv, anti-VEGF-D-scFv, anti- VEGF-E-scFv, or anti-VEGF-F-scFv, or a combination thereof)] conjugated to the nanoparticle, wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm) (e.g., wherein the AAINC has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) for use in a method of in vivo diagnosis of a disease or condition in a subject, wherein the in vivo diagnosis comprises: delivering the AAINC to the subject; and detecting (e.g., via PET, X-ray, MRI, CT, etc.) the imaging agent.

[0032] In another aspect, the invention is directed to a treatment comprising: (a) a therapeutically effective amount of the nanoparticle conjugate for use in combination with one or more of the following: (1) an anti-angiogenic agent, (2) a radiotherapeutic agent, (3) a chemotherapeutic agent, (4) an immunotherapeutic agent, or (5) engineered cells (e.g., CAR T cells), or (b) a therapeutically effective amount of one or more of the following: (1) an anti-angiogenic agent, (2) a radiotherapeutic agent, (3) a chemotherapeutic agent, (4) an immunotherapeutic agent, or (5) engineered cells (e.g., CAR T cells) for use in combination with the nanoparticle conjugate, for use in a method of treating a disease or condition (e.g., inflammation, e.g., neuroinflammation, e.g., edema, e.g., radiation necrosis, e.g., cancer, e.g., breast cancer, e.g., brain cancer, e.g., breast cancer brain - 14 - 12799878v1Attorney Docket No. 2018488-0014 metastases, e.g., a combination of any one inflammation, neuroinflammation, edema, radiation necrosis, cancer, breast cancer, brain cancer, or breast cancer brain metastases) and / or preventing disease or condition occurrence or recurrence in a subject and / or mitigating the onset and / or progression of a disease (e.g., wherein the immunotherapeutic and / or engineered cells comprise(s) an engineered immunotherapy [e.g., for localized treatment, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid primary tumors, e.g., metastatic tumors), brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, or other diseases] comprising: an engineered immune cell [e.g., a macrophage, e.g., a neutrophil, e.g., a T cell (e.g., a Steap1 CAR-T cell), e.g., an NK cell] comprising (a) a chimeric antigen receptor (CAR) and (b) a bispecific immune cell engager (e.g., a bispecific T-cell engager or “BiTE”, e.g., a bispecific macrophage engager, e.g., a bispecific neutrophil engager)) (e.g., wherein the immunotherapeutic and / or engineered cells comprise(s) an engineered immunotherapy [e.g., for localized treatment, e.g., for combinatorial therapy, e.g., for treatment of cancer (e.g., solid primary tumors, e.g., metastatic tumors), brain injury (e.g., radiation, trauma), neurological disorders, inflammatory diseases, or other diseases] comprising: an engineered immune cell comprising a CAR T cell comprising one or more targeting ligands, e.g., a MUC16- targeting ligand).

[0033] Elements of embodiments involving one aspect of the invention (e.g., methods) can be applied in embodiments involving other aspects of the invention (e.g., systems), and vice versa. DEFINITIONS

[0034] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definition for the following terms and other terms are set forth throughout the specification.

[0035] Administration: As used herein, the term “administration” typically refers to the administration of a composition comprising a nanoparticle to a subject or system. In general, any route of administration may be utilized including, for example, parenteral (e.g., intravenous), oral, topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into body compartments. In some embodiments, administration is oral. Additionally or alternatively, in some embodiments, administration is parenteral. In some embodiments, administration is intravenous. In some embodiments, administration is intraperitoneal. - 15 - 12799878v1Attorney Docket No. 2018488-0014

[0036] Agent: The term “agent”, as used herein, may refer to a compound, molecule, or entity of any chemical and / or biological class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety. In some embodiments, the term may refer to a nanoparticle.

[0037] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kD tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about to or less than 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure. Each heavy chain is comprised of at least four domains (each about 110 amino acids long)– an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CH1, CH2, and the carboxy-terminal CH3 (located at the base of the Y’s stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain is comprised of two domains – an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another “switch”. Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the - 16 - 12799878v1Attorney Docket No. 2018488-0014 domains, and the CDR loop regions from both the heavy and light chains are brought together in three- dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. The Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity. As is known in the art, affinity and / or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and / or utilized in accordance with the present disclosure include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation. For purposes of the present disclosure, In some embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and / or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art. Moreover, the term “antibody” as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, an antibody utilized in accordance with certain embodiments of the present disclosure is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiment, an antibody is an engineered antibody. - 17 - 12799878v1Attorney Docket No. 2018488-0014

[0038] Antibody agent: As used herein, the term “antibody agent” refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc, as is known in the art. In many embodiments, the term “antibody agent” is used to refer to one or more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, embodiments, an antibody agent utilized in accordance with certain embodiments of the present disclosure is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload or other pendant group). In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it - 18 - 12799878v1Attorney Docket No. 2018488-0014 shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.

[0039] Antigen: The term “antigen”, as used herein, refers to an agent that elicits a biological response and / or an agent that is bound to a binding agent (or “binder”). In some embodiments, an antigen elicits an angiogenic and / or immune response. In some embodiments, an antigen is VEGF (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, or VEGF-F). In some embodiments, an antigen is HER2. In some embodiments, an antigen is CD3ε. In some embodiments, an antigen is EGFR. In some embodiments, an antigen is a4-1BB. In some embodiments, an agent is EpCam. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies); in some embodiments, an agent elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen, e.g., involving angiogenesis). In some embodiments, an - 19 - 12799878v1Attorney Docket No. 2018488-0014 antigen binds to an antibody and may or may not induce a particular physiological response in an organism.

[0040] Biocompatible: The term “biocompatible”, as used herein, refers to materials that do not cause significant harm to living tissue when placed in contact with such tissue, e.g., in vivo. In some embodiments, materials are “biocompatible” if they are not toxic to cells. In some embodiments, materials are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death. In some embodiments, materials are biodegradable.

[0041] Cancer: As used herein, the term “cancer” refers to a malignant neoplasm or tumor (Stedman’s Medical Dictionary, 25th ed.; Hensly ed.; Williams & Wilkins: Philadelphia, 1990). Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi’s sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett’s adenocarcinoma); Ewing’s sarcoma; eye cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B cell ALL, T cell ALL), acute myelocytic leukemia (AML) (e.g., B cell AML, T cell AML), chronic myelocytic leukemia (CML) (e.g., B cell CML, T cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B cell CLL, T cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B cell HL, T cell HL) and non Hodgkin lymphoma (NHL) (e.g., B cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic - 20 - 12799878v1Attorney Docket No. 2018488-0014 lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B cell lymphomas (e.g., mucosa associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B cell lymphoma, splenic marginal zone B cell lymphoma), primary mediastinal B cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (e.g., Waldenstrom’s macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T cell NHL such as precursor T lymphoblastic lymphoma / leukemia, peripheral T cell lymphoma (PTCL) (e.g., cutaneous T cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T cell lymphoma, extranodal natural killer T cell lymphoma, enteropathy type T cell lymphoma, subcutaneous panniculitis like T cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms’ tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget’s disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, - 21 - 12799878v1Attorney Docket No. 2018488-0014 malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget’s disease of the vulva). In some embodiments, cancer is a breast cancer brain metastases (BCBM).

[0042] Chemotherapeutic Agent: As used herein, the term “chemotherapeutic agent” or “oncolytic therapeutic agent”(e.g., anti-cancer drug, e.g., anti-cancer therapy, e.g., immune cell therapy) has its art-understood meaning referring to one or more pro-apoptotic, cytostatic and / or cytotoxic agents, and / or hormonal agents, for example, specifically including agents utilized and / or recommended for use in treating one or more diseases, disorders or conditions associated with undesirable cell proliferation. In many embodiments, chemotherapeutic agents and / or oncolytic therapeutic agents are useful in the treatment of cancer. In some embodiments, a chemotherapeutic agent and / or oncolytic therapeutic agents may be or comprise one or more hormonal agents (e.g., androgen inhibitors), one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g., microtubule targeting agents such as taxanes, maytansine and analogs thereof, of), one or more epothilones, one or more histone deacetylase inhibitors HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of one or more of the following (i.e., that share a relevant anti-proliferative activity). In some particular embodiments, a chemotherapeutic agent may be or comprise one or more of Actinomycin, all-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and / or analogs thereof (e.g., DM1) Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof. In some embodiments, a chemotherapeutic agent may be utilized in the context of an antibody-drug conjugate. In some - 22 - 12799878v1Attorney Docket No. 2018488-0014 embodiments, a chemotherapeutic agent is one found in an antibody-drug conjugate selected from the group consisting of: hLLl -doxorubicin hRS7-SN-38, hMN-l4-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLLl-SN-38, hRS7- Pro-2-P-Dox, hMN-l4-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20- Pro-2-P-Dox, hPAM4-Pro-2-PDox, hLLl-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA , antibody-drug conjugate (ADC), RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG- 7600, RG-7636, ABT-414, IMGN-853, IMGN-529, vorsetuzumab mafodotin, and lorvotuzumab mertansine. In some embodiments, a chemotherapeutic agent may be or comprise one or more of famesyl-thiosalicylic acid (FTS), 4- (4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), estradiol (E2), tetramethoxystilbene (TMS), δ-tocatrienol, salinomycin, or curcumin. In some embodiments, chemotherapeutic agents and / or oncolytic therapeutic agents for anti-cancer treatment comprise (e.g., are) biological agents such as tumor-infiltrating lymphocytes, CAR T-cells, antibodies, antigens, therapeutic vaccines (e.g., made from a patient’s own tumor cells or other substances such as antigens that are produced by certain tumors), immune-modulating agents (e.g., cytokines, e.g., immunomodulatory drugs or biological response modifiers), checkpoint inhibitors) or other immunologic / pharmacologic agents (e.g., PI3K^-selective inhibitor targeting myeloid cells or IPI- 549). In some embodiments, immunologic agents include immunoglobins, immunostimulants (e.g., bacterial vaccines, colony stimulating factors, interferons, interleukins, therapeutic vaccines, vaccine combinations, viral vaccines) and / or immunosuppressive agents (e.g., calcineurin inhibitors, interleukin inhibitors, TNF alpha inhibitors). In some embodiments, hormonal agents include agents for anti- androgen therapy (e.g., Ketoconazole, ABiraterone, TAK-700, TOK-OOl, Bicalutamide, Nilutamide, Flutamide, Enzalutamide, ARN-509).

[0043] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those - 23 - 12799878v1Attorney Docket No. 2018488-0014 adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0044] Radiolabel: As used herein, “radiolabel” refers to a moiety comprising a radioactive isotope of at least one element. Exemplary suitable radiolabels include but are not limited to those described herein. In some embodiments, a radiolabel is one used in positron emission tomography (PET). In some embodiments, a radiolabel is one used in single-photon emission computed tomography (SPECT). In some embodiments, radioisotopes comprise99mTc,111In,64Cu,67Ga,186Re,188Re,153Sm,177Lu,67Cu,123I,124I,125I,11C,43N,150 ,18F,161Ho,149Pm,90Y,213Bi,103Pd,159Gd,140La,198Au,199Au,169Yb,175Yb,165Dy,166Dy,67Cu,105Rh,111Ag,225Ac,192Ir, and89Zr.

[0045] Subject: As used herein, the term “subject” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, subjects are mammals, particularly primates, especially humans. In some embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In some embodiments (e.g., particularly in research contexts) subject mammals are, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like.

[0046] Therapeutically effective amount: as used herein, is meant an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not in fact require successful treatment be achieved in a particular individual. Rather, a - 24 - 12799878v1Attorney Docket No. 2018488-0014 therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen.

[0047] Therapeutic agent: As used herein, the phrase “therapeutic agent” in general refers to any agent that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject.

[0048] Treatment: As used herein, the term “treatment” (also “treat” or “treating”) refers to administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition.

[0049] Tumor: As used herein, the term “tumor” refers to an abnormal growth of cells or tissue. In some embodiments, a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments as discussed herein, a tumor is associated with, or is a manifestation of, a cancer. In some embodiments as discussed herein, a tumor may be a solid tumor.

[0050] Drawings are presented herein for illustration purposes, not for limitation. - 25 - 12799878v1Attorney Docket No. 2018488-0014 BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 is an illustrative schema for screening and identification of nanoparticle conjugates as described herein.

[0052] FIG. 2 is an illustrative schema for Combinatorial Treatment Plan for Murine and Human Breast Cancer Brain Metastases (BCBMs)t.

[0053] FIG. 3 is an illustrative schema for Radiation Necrosis Treatment Plan for Murine BCBM.

[0054] FIG. 4 is a flowchart describing steps relating to designed ankyrin repeat protein (DARPin) selection method.

[0055] FIG. 5 shows SDS-PAGE results of single- and dual-targeting DARPins.

[0056] FIG. 6 summarizes head-to-head comparison of different aVEGF proteins.

[0057] FIG. 7 is an illustrative schema for aVEGF DARPin binding affinity SPR assay.

[0058] FIG. 8 is an illustrative schema for Cornell Prime Dot-DARPin Drug-Conjugate (CxD).

[0059] FIG. 9 shows an image of Therapeutic Index Enhancer (TIE).

[0060] FIG. 10 is an illustrative schema for Multi-specific CxD concept.

[0061] FIG. 11 represents selected binders for CxD Program.

[0062] FIG. 12 is an illustrative schema for proprietary binders for CxD program achieved via Phage Display-Based DARPin Binder Library Generation and Affinity Screening.

[0063] FIG. 13A is an illustrative schema for mapping the pET28a(+) expression vector containing the HER2×VEGF bispecific DARPin gene (6079 bp) under the T7 promoter, with an N- terminal 6×His-tag and tobacco etch virus (TEV) protease cleavage site for affinity purification, and a C-terminal (G3S)2linker followed by a cysteine residue for site-specific conjugation.

[0064] FIG. 13B shows annotated amino acid sequence of the expressed HER2×VEGF bispecific DARPin. - 26 - 12799878v1Attorney Docket No. 2018488-0014

[0065] FIG. 13C shows SEC profiles of the purified HER2×VEGF bispecific DARPin, monospecific anti-HER2, and anti-VEGF monospecific DARPins, compared against protein molecular weight standards.

[0066] FIG. 13D shows SDS-PAGE analysis of purified DARPins showing molecular weights for bispecific HER2×VEGF DARPin, monospecific anti-HER2 and anti-VEGF DARPins, and evaluation before and after reducing treatments with 1 mM tris(2-carboxyethyl)phosphine (TCEP) or β- mercaptoethanol (BME).

[0067] FIG. 14 is an illustrative schema for potential applications for HER2×VEGF bispecific DARPin-azide through orthogonal click chemistry bioconjugation. The azide-functionalized HER2×VEGF bispecific DARPin can undergo SPAAC click chemistry reactions with diverse dibenzocyclooctyne (DBCO)-modified payloads, including (1) fluorescent dyes for optical imaging (e.g., BP Fluor 647-DBCO), (2, and 3) radioisotope chelators for PET / SPECT (single photon emission computed tomography) imaging or radiotherapy (e.g., deferoxamine-DBCO and DOTA-PEG5-C6- DBCO), (4) cleavable cytotoxic drug linkers (e.g., DBCO-PEG4-Val-Cit-PAB-MMAE) for targeted therapy, and (5) DBCO-functionalized nanoparticles (e.g., Au, SiO2nanoparticles) for advanced nanomedicine delivery platforms.

[0068] FIG. 15 shows plasmid design and amino acid sequences of bispecific HER2×VEGF DARPin and monospecific anti-HER2 and anti-VEGF DARPins. (a) Map of the pET28a(+) vector encoding the HER2×VEGF bispecific DARPin (6079 bp) under the T7 promoter, featuring an N- terminal 6×His-tag and TEV cleavage site for affinity purification, a (G₄S)₃ linker, and a C-terminal cysteine for site-specific conjugation, (b) Amino acid sequence of the HER2×VEGF bispecific DARPin, showing the modular structure: 6×His-tag-TEV site, anti-HER2 DARPin domain, (G₄S)₃ linker, anti-VEGF DARPin domain, and C-terminal linker-cysteine, (c) Map of the pET28a(+) vector encoding the anti-HER2 monospecific DARPin (5656 bp), similarly organized with a 6×His-tag-TEV site and a C-terminal cysteine linker, (d) Amino acid sequence of the anti-HER2 monospecific DARPin, highlighting the anti-HER2 DARPin domain and C-terminal cysteine linker, (e) Map of the pET28a(+) vector encoding the anti-VEGF monospecific DARPin (5656 bp), containing a 6×His-tag- TEV site and a C-terminal cysteine linker for downstream functionalization, and (f) Amino acid - 27 - 12799878v1Attorney Docket No. 2018488-0014 sequence of the anti-VEGF monospecific DARPin, showing the anti-VEGF DARPin domain and C- terminal cysteine linker.

[0069] FIG. 16 is an illustrative workflow schema for the expression, purification, and characterization of the HER2×VEGF bispecific DARPin.

[0070] FIG. 17A is an illustrative schema for the10-fold scale-up from 30 mL (1× scale) to 300 mL (10× scale) bacterial expression cultures.

[0071] FIG. 17B shows SDS-PAGE results for purified HER2×VEGF bispecific DARPins under non-reducing and reducing conditions, comparing 1× and 10× scale productions.

[0072] FIG. 18 shows SEC profiles of HER2×VEGF bispecific DARPin-Cysteine that were analyzed at Day 0, Day 4, Day 7, Day 14, and Day 21, under storage at 4^°C (solid lines) and 25^°C (dotted lines).

[0073] FIG. 19 shows SDS-PAGE analysis of HER2×VEGF bispecific DARPin-Cysteine stability after 21-day storage. (a) SDS-PAGE analysis results under non-reducing and reducing conditions, (b) Quantitative results of monomer and dimer content of (a).

[0074] FIG. 20 shows the result of circular dichroism (CD) thermal denaturation analysis of HER2×VEGF bispecific DARPin-Cysteine.

[0075] FIG. 21 shows results from flow cytometry analysis of (a) NCI-N87, (b) BT-474, and (c) MDA-MB-231 cells stained with anti-HER2 PE-mAb or control PE-mAb, compared to unstained controls.

[0076] FIG. 22 shows results from flow cytometry analysis of DARPin binding to HER2- positive (NCI-N87, BT-474) and HER2-negative (MDA-MB-231) breast cancer cell lines.

[0077] FIG. 23 is an illustrative schema for the anti-VEGF ELISA assay setup. (1) Wells are pre-coated with recombinant human 165-amino acid isoform of VEGF-A (VEGF165) protein, (2) His- tagged DARPins (HER2×VEGF bispecific or monospecific controls) bind specifically to the immobilized VEGF165antigen, (3) Biotinylated anti-HisTag antibody recognizes the bound DARPins, and (4) streptavidin-HRP binding and catalysis of a colorimetric reaction using 3,3′,5,5′- tetramethylbenzidine (TMB) substrate for detection. - 28 - 12799878v1Attorney Docket No. 2018488-0014

[0078] FIG. 24 represents results from ELISA analysis of different proteins.

[0079] FIG. 25A is an illustrative schema for the conjugation strategy: the free thiol group of the C-terminal cysteine in HER2×VEGF bispecific DARPin-Cysteine was selectively modified with Azide-PEG3-Maleimide through a thiol–maleimide coupling reaction, generating HER2×VEGF bispecific DARPin-Azide.

[0080] FIG. 25B shows SEC profiles monitoring the stability of HER2×VEGF bispecific DARPin-Azide over 23 days at 4^°C (solid lines) and 25^°C (dotted lines).

[0081] FIG. 25C shows quantitative comparison of dimer content during storage at 25^°C, highlighting the substantial suppression of dimerization following Azide-PEG3 modification, in contrast to the progressive dimer accumulation seen with the unmodified DARPin-Cysteine.

[0082] FIG. 26 shows SEC-HPLC results relating to purified HER2×VEGF bispecific DARPin-azide.

[0083] FIG. 27 shows SEC-HPLC results relating to conjugation of HER2×VEGF bispecific DARPin-azide to different DBCO-functionalized moieties.

[0084] FIG. 28 shows SDS-PAGE results relating to HER2×VEGF bispecific DARPin and its bioconjugates under (a) non-reducing and (b) reducing conditions.

[0085] FIG. 29 shows SEC-HPLC dual-wavelength analysis confirming covalent conjugation of BP Fluor 647 to HER2×VEGF bispecific DARPin.

[0086] FIG. 30 shows SPR assay development and validation for monospecific DARPins. (a) Schematic of SPR setup with HER2 antigen immobilized on a carboxymethylated dextran sensor chip (CM5) and anti-HER2 DARPin as the analyte, (b) Sensorgrams showing binding of anti-HER2 DARPin to HER2-immobilized chip, (c) Sensorgrams showing no detectable interaction with VEGF165- immobilized chip, (d) Kinetic parameters (kon, koff, and KD) for anti-HER2 DARPin interaction with HER2, (e) Schematic of SPR setup with VEGF165 antigen immobilized on a CM5 chip and anti-VEGF DARPin as the analyte, (f) Sensorgrams showing no detectable binding of anti-VEGF DARPin to HER2-immobilized chip, (g) Sensorgrams showing specific interaction with VEGF165-immobilized chip, and (h) Kinetic parameters (kon, koff, and KD) for anti-VEGF DARPin interaction with VEGF165. - 29 - 12799878v1Attorney Docket No. 2018488-0014

[0087] FIG. 31 shows SPR characterization of dual-target binding kinetics of functionalized HER2×VEGF bispecific DARPin bioconjugates. (a) Schematic overview of the SPR setup showing dual flow channels with immobilized HER2 (left, Channel #1) and VEGF165 (right, Channel #2) on separate CM5 sensor chip surfaces, (b–d) Characterization of HER2×VEGF bispecific DARPin–BP Fluor 647 conjugate: (b) Sensorgram showing binding to HER2; (c) Schematic of HER2×VEGF bispecific DARPin–BP Fluor 647 conjugate; (d) Sensorgram showing binding to VEGF165, (e–g) DOTA-conjugated HER2×VEGF bispecific DARPin, (h–j) DFO-conjugated HER2×VEGF bispecific DARPi, and (k–m) MMAE cleavable linker–conjugated HER2×VEGF bispecific DARPin. For each bioconjugate, sensorgrams demonstrate preserved dual-target binding post-conjugation, with corresponding KD values reported for HER2 and VEGF165 (c, f, i, l).

[0088] FIG. 32 shows SPR validation of unmodified and azide-modified HER2×VEGF bispecific DARPin binding to HER2 and VEGF165. (a) Schematic of dual-channel SPR setup using CM5 sensor chip immobilized with HER2 (left) and VEGF165 (right), (b–d) Sensorgrams of HER2×VEGF bispecific DARPin-Cysteine binding HER2 and VEGF165, and (e–g) Sensorgrams of HER2×VEGF bispecific DARPin-azide binding HER2 and VEGF165.

[0089] FIG. 33 shows SEC profiles of HER2×EGFR bispecific DARPin, CD3×HER2 bispecific DARPin, anti-EGFR monospecific DARPin, HER2×VEGF bispecific DARPin, anti-HER2 monospecific DARPin, anti-VEGF monospecific DARPin, and Protein standard.

[0090] FIG. 34 is an illustrative schema representing steps of AI-driven computer-aided protein design.

[0091] FIG. 35 is an illustrative schema for CD3xHER2 bispecific DARPin–C’ dot conjugate as a T cell engager for immunotherapy.

[0092] FIG. 36 is an illustrative schema for HER2×VEGF Bispecific DARPin– Radiotherapeutic Conjugate.

[0093] FIG. 37 shows results from SDS-PAGE analysis of Anti-VEGF scFv at different development stages.

[0094] FIG. 38 shows results from SDS-PAGE analysis of Anti-MUC16 scFv at different development stages. - 30 - 12799878v1Attorney Docket No. 2018488-0014

[0095] FIG. 39 shows results from SDS-PAGE analysis of Anti-CD3 scFv at different development stages.

[0096] FIG. 40 shows an implementation of a network environment for use in providing systems, methods, and architectures according to an exemplary embodiment of the present disclosure.

[0097] FIG. 41 shows an example of a computing device and a mobile computing device that can be used to implement the techniques according to an exemplary embodiment of the present disclosure. DETAILED DESCRIPTION

[0098] It is contemplated that methods, compositions, and processes of the claimed disclosure encompass variations and adaptations developed using information from the embodiments described herein. Adaptation and / or modification of the methods, compositions, and processes described herein may be performed, as contemplated by this description.

[0099] Throughout the description, where methods, compositions, and processes are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited steps.

[0100] It should be understood that the order of steps or order for performing certain action is immaterial so long as the disclosure remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0101] The mention herein of any publication, for example, in the Background section, is not an admission that the publication serves as prior art with respect to any of the claims presented herein. The Background section is presented for purposes of clarity and is not meant as a description of prior art with respect to any claim. Documents are incorporated herein by reference as noted. Where there is any discrepancy in the meaning of a particular term, the meaning provided in the Definition section above is controlling. Headers are provided for the convenience of the reader – the presence and / or placement of a header is not intended to limit the scope of the subject matter described herein. Brain metastases and current need - 31 - 12799878v1Attorney Docket No. 2018488-0014

[0102] The brain tumor microenvironment (TME), along with the evolution of growth-induced stresses, play pivotal roles in facilitating invasion, metastasis, and inflammatory responses, in turn, leading to peritumoral edema, mass effect, and neurological sequelae, such as headache, seizures and brain herniation. A key driver of brain metastasis rests on the reciprocal TME interactions occurring among invading breast cancer cells and astrocytes, microglia, brain endothelial cells, as well as resident and infiltrating immune cells. Alterations in BBB permeability, as well as tumor intrinsic factors, including angiogenesis, hypoxia, DNA damage repair, and neuroinflammation, may be triggered in response to ensuing tissue damage, aiding formation of metastases. The suppressive TME of BCBMs also promotes disease through downregulation of immune-related pathways and upregulation of tumor intrinsic factors. Poor clinical outcomes have been associated with decreased cytotoxic CD8+ T cells and M1 proinflammatory macrophages, along with increased pro-tumorigenic M2 macrophages. These results underscore a crucial and timely need to develop new and effective targeted therapeutic strategies for the early prevention and treatment of inflammation and BCBMs. The application of advanced neuroimaging paradigms, such as multiparametric magnetic resonance imaging (MRI), for the early detection of disease and neuroinflammation (e.g., edema), along with syngeneic preclinical BCBM models that better capture TME heterogeneity are critically needed to fill current gaps supporting such developments.

[0103] Moreover, BCBM poses a significant clinical challenge, particularly in human epidermal growth factor receptor 2 (HER2)-positive cases. Despite improvements in systemic HER2- targeted therapies, including trastuzumab, pertuzumab, and small-molecule inhibitors such as tucatinib, their efficacy in the brain is often limited by inadequate penetration across the blood-brain barrier (BBB) and the emergence of resistance mechanisms. The recent introduction of trastuzumab deruxtecan (T-DXd, Enhertu), a HER2-targeting antibody-drug conjugate (ADC), has shown promising efficacy in HER2-positive metastatic breast cancer, including patients with brain metastases. However, its large molecular size (~150 kDa) limits efficient BBB penetration, and its clinical use is complicated by significant risks, including interstitial lung disease (ILD). Additionally, tumor-driven angiogenesis, largely mediated by vascular endothelial growth factor (VEGF), plays a critical role in BCBM progression and resistance to HER2-targeted therapies. Anti-VEGF therapies, such as bevacizumab, - 32 - 12799878v1Attorney Docket No. 2018488-0014 have been used to reduce peritumoral edema and normalize vasculature, improving drug delivery. However, VEGF blockade alone is insufficient for controlling disease progression in BCBM.

[0104] Radiotherapy (RT), used as a standard treatment for BCBMs to achieve local control and reduce recurrence, can also remodel the TME and enhance BBB permeability for drug delivery. In addition to activating adaptive and innate immune systems through DNA damage responses and direct tumor cell kill, it generates mutations in tumor-specific peptides (i.e., neoantigens) and promotes localized and systemic inflammatory reactions that increase immune cell trafficking. These properties offer the opportunity to develop new combination strategies that synergize with new technologies (e.g., nanomedicines) to regulate BCBM growth, the suppressive TME, and associated inflammatory changes. Only a paucity of BCBM combinatorial studies have been conducted with RT and nanomaterials, but utilized immunodeficient models for generating intracranial xenografts, precluding assessment of the TME. Dose-limiting complications of RT, such as the onset of radiation necrosis (RN) can arise, typically occurring 6 to 24 months post-SRS treatment in ~10% of BCBM patients. The emergence of RN is thought to be the result of radiation damage to cell populations within the TME (e.g., endothelial and glial cells), with the release of inflammatory mediators, such as hypoxiainducible factor 1a (HIF-1a) and vascular endothelial growth factor (VEGF). Inflammatory side effects in cancer subjects and current need

[0105] Enhanced angiogenesis supports early metastatic disease spread and is associated with highly proliferative breast tumors, such as triple negative breast cancer (TNBC), which express high levels of the angiogenic protein, VEGF. VEGF-A, a well-characterized factor, binds to tyrosine kinase receptors, VEGFR1 and VEGFR2, and regulates key processes throughout the angiogenic cascade, as well as partially mediates loss of BBB integrity. The vascular remodeling factor, VEGF-B, operates through a VEGF-A independent mechanism, binding only to VEGFR1 to promote metastatic disease. Inhibition of VEGF-B may therefore serve as an alternative approach for developing new therapies to treat metastases. Anti-angiogenesis inhibitors of VEGFR tyrosine kinase activity and neutralizing antibodies (e.g., bevacizumab / Avastin®) target VEGF signaling pathways with the potential to restore vascular integrity, decrease permeability, and reduce edema. Although many approved agents have effectively managed inflammatory changes, edema, and distant metastases, clinical benefits are short- lived, limited by adverse events, reduced efficacy, tumor recurrence, and acquired drug resistance. - 33 - 12799878v1Attorney Docket No. 2018488-0014 Alternative VEGF-targeting nano delivery systems have been developed for treating BCBMs, but are sparse, with no candidates advancing to clinical trials. While a few studies showed efficacy in xenografted BCBM models, the large particle sizes (greater than 40 nanometer (nm)) and lack of TME and toxicology assessments limited evaluation of product translation. The technology developments described herein overcome these limitations and transform the landscape of treatments for BCBM. Ovarian cancer and current need

[0106] Moreover, ovarian cancer (OC) is a leading cause of mortality in women with gynecological malignancies, with more than 70% of patients presenting with advanced disease. The emergence of many therapeutic modalities to treat advanced / refractory disease has generally shown only limited benefit. Recently, chimeric antigen receptor (CAR) T-cell therapies, have been designed to target mucin-16 (MUC16, CA125) and mesothelin (MSLN), glycoproteins overexpressed on OC cells that promote tumor progression. Their effectiveness, however, has been limited, largely attributable to suppressive tumor microenvironments (TME) that drive mutational resistance, T-cell exhaustion, a lack of T-cell persistence, and other factors. These impediments highlight the importance of advancing new efficacious therapies that, in combination with CAR T-cells, improves the performance of immunotherapies (IT) and sustains treatment responses through reversal of suppressive activities. A safe, clinically validated, and therapeutic ultrasmall (less than 8 nm) fluorescent silica particle, Cornell prime dots (C’ dots) provides one such solution. Without an attached cytotoxic drug, unmodified C’ dots act as an adjuvant-therapy, enhancing immunogenicity, cytotoxicity, and IT effectiveness over controls in solid TMEs.

[0107] Although early management of advanced OC with cytoreductive surgery and cytotoxic drugs are effective, mortality rates remain high due to disease relapse. The use of CAR T cells in the context of solid tumor malignancies is evolving; however, immune-based therapies for OC are compromised by a suppressive TME that inhibits their function. For OC, a series of targets, including MSLN and MUC1 (e.g., retained extracellular domain, MUC16ecto) have been described and shown promising preclinical data. However, despite ongoing CAR T cell trials for advanced / refractory OC, clinical efficacy has been modest. It is for this reason that new combinatorial partners exhibiting diverse and potent anti-tumor activities are urgently needed to enhance treatment efficacy and improve T cell function by reversing TME suppressive activities. More recently, studies have reported that - 34 - 12799878v1Attorney Docket No. 2018488-0014 strategies exploiting endogenous ligand-receptor interactions in the construction of CAR T therapies can be highly effective. To this end, MUC16-targeting MSLN-CAR T cells have been developed (referred to herein as MSLN-CAR T cells) that exploit the binding capacity of mesothelin to MUC16 through the fusion of human MSLN to 2nd generation human intracellular signal domains CD28 and CD3z. Clinical trials evaluating alternative IT regimens are presently active, however no single approved product has yet emerged. The goals of such studies are distinctly different from those using emerging 'self-therapeutic' nanomedicine strategies20. In the present disclosure, the goal is reducing suppressive activities, improve CAR T cell performance and efficacy, and limit adverse events. Most nanomedicines exhibit limitations for translation due to their relatively large size (e.g., greater than 30 nm), making them highly prone to off-target accumulations. The favorable PK of C’ dots address such limitations, allowing them to serve as effective adjuvant therapies for TME modulation. To enhance specificity, C’ dots can be conjugated with new potent antibody mimetic protein therapies, DARPins, that target the retained portion of MUC16ecto and MSLN.

[0108] The treatment of relapsed / refractory OC with CAR T cell therapy has demonstrated only modest clinical outcomes, largely the result of a suppressive TME that inhibits the function of tumor- specific effector cells, a lack of effector T cell persistence, and target antigen escape. Overcoming these limitations to improve treatment efficacy is urgently needed and requires new mechanistically- driven combinatorial partners that can modulate an immune suppressive TME to one that is pro- inflammatory, augment CAR T cell expansion and function, as well as recruit endogenous anti-tumor immune effectors targeted to a broader array of tumor cell antigens and neo-antigens. Harnessing the adjuvant-therapeutic properties of C’ dots, reductions in key suppressive T / myeloid cell populations / receptors and cytokines, activation of innate / effector immune cells, upregulation of proinflammatory signatures, and induction of cell death programs (e.g., ferroptosis) were previously observed in multiple solid tumors following a multi-dose regimen administered systemically. These effects additionally led to a survival benefit in combination with ITs.

[0109] Bispecific antibodies targeting HER2, and VEGF have been explored to address these challenges. YY0411, an IgG-Decoy receptor bispecific antibody, has demonstrated significant tumor inhibition in preclinical lung cancer models by effectively neutralizing HER2 signaling and sequestering VEGF. However, it was not specifically designed for BCBM, and its large size may limit - 35 - 12799878v1Attorney Docket No. 2018488-0014 penetration into brain metastases. Additionally, antibody-based bispecifics often face challenges in manufacturing complexity, stability, and aggregation, as well as the need for fragment crystallizable region (Fc) modifications to improve their pharmacokinetics and avoid immune effector function complications. Designed Ankyrin Repeat Proteins (DARPins) offer a promising alternative due to their small size (~14 - 18 kDa per domain), high stability, modularity, and cost-effective bacterial production.

[0110] DARPins are engineered scaffold proteins based on naturally occurring ankyrin repeat motifs, which provide a rigid framework for high-affinity molecular recognition. Unlike antibodies, DARPins lack disulfide bonds, making them exceptionally stable even under harsh conditions, and their simple structure allows for facile engineering into multi specific formats. These properties make DARPins attractive for therapeutic applications requiring enhanced tissue penetration, improved stability, and reduced immunogenicity. Nanoparticle conjugates

[0111] The present disclosure provides for nanoparticle conjugates. Disclosed herein are high- affinity Designed Ankyrin Repeat Protein (DARPin)-based nanoparticle conjugates. Also disclosed herein are anti-angiogenic and / or immunomodulatory nanoparticle conjugates (“AAINCs”). For example, the present disclosure describes nanoparticle conjugates that simultaneously targeting tumor cell surface receptors and soluble growth factors offers a powerful approach to enhance treatment specificity and overcome resistance in cancer therapy.

[0112] Among other things, presented herein is the development of a modular bispecific Designed Ankyrin Repeat Protein (DARPin) binder that targets both human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor (VEGF), two clinically relevant cancer- associated antigens. The described construct(s) were engineered with a C-terminal cysteine to allow site-specific chemical modification through bioorthogonal click chemistry. Following efficient expression in Escherichia coli and high-purity isolation, the DARPin binder was converted into an azide-functionalized format and conjugated to a diverse set of functional payloads. These included fluorescent dyes for optical imaging, radionuclide chelators for diagnostic and therapeutic isotope labeling, and a cleavable cytotoxic drug linker for targeted chemotherapy. Each conjugate was confirmed to be monodisperse and high purity through comprehensive analytical characterization. - 36 - 12799878v1Attorney Docket No. 2018488-0014 Binding kinetics measured by surface plasmon resonance demonstrated that all functionalized constructs retained strong and specific affinity to both HER2 and VEGF, with dissociation constants in the low picomolar to nanomolar range. Importantly, site-specific conjugation had minimal impact on antigen recognition, underscoring the structural resilience of the binding domains. The present disclosure establishes a flexible and scalable bispecific DARPin scaffold that supports efficient, plug- and-play conjugation of imaging and therapeutic payloads without compromising function. The preserved dual-targeting capability across a wide range of modifications positions this platform as a promising foundation for precision oncology tools, including image-guided diagnostics, targeted radiotherapy, and site-specific drug delivery.

[0113] As described herein, a bispecific HER2×VEGF DARPin, integrating high-affinity inhibition of both targets into a single, compact molecule improved for therapeutic application, was developed. To achieve effective dual targeting, a bispecific DARPin was designed by incorporating DARPin G3, a well-characterized high-affinity HER2 binder, and Abicipar pegol, the first clinically developed anti-VEGF DARPin. These domains were linked using the widely used flexible (G4S)₃ linker, ensuring proper orientation and function of both binding sites. The HER2×VEGF bispecific DARPin’s dual-target binding kinetics, biochemical stability, and structural integrity are systematically characterized through a comprehensive suite of biophysical, biochemical, and functional assays. The goal is to establish a scalable, highly stable bispecific protein with the potential for improved therapeutic efficacy compared to existing antibody-based strategies. Beyond its design and characterization, a click chemistry-based bioconjugation strategy for site-specific functionalization, allowing controlled labeling with imaging probes, half-life extension moieties, or therapeutic payloads without compromising stability, is further developed.

[0114] A HER2×VEGF bispecific DARPin featuring a modular C-terminal cysteine for site- specific azide functionalization and orthogonal click chemistry conjugation was successfully designed, engineered, and characterized herein. The construct was efficiently expressed in E. coli, yielding high monomeric purity across scalable production formats. Bioconjugation with diverse DBCO- functionalized moieties—including fluorescent dyes, radiometal chelators, and a cleavable MMAE drug linker—was achieved with high efficiency and preserved protein integrity, as confirmed by SEC-HPLC and SDS-PAGE. SPR analyses demonstrated that all conjugated variants retained strong, dual-specific - 37 - 12799878v1Attorney Docket No. 2018488-0014 binding to HER2 and VEGF165, with KDvalues in the picomolar to low nanomolar range. The present disclosure established a robust and flexible DARPin-based platform that allows the generation of multifunctional bioconjugates for targeted imaging and therapy. The preserved high-affinity dual- targeting following conjugation supports broad biomedical utility—from optical imaging and PET / SPECT diagnostics to targeted radiotherapy and drug delivery. The modular design provides a foundation for rapid development of next-generation, dual-functional DARPins in precision oncology, offering new opportunities for tumor-targeted diagnostics, therapeutics, and combination treatment strategies.

[0115] The present disclosure also describes an exemplary development and evaluation of an efficacious AIINC (e.g., an ultrasmall VEGF-targeting particle adjuvant) that can suppress inflammation-mediated processes / edema and growth in BCBM models – serving as a safe alternative to corticosteroids. Towards this end, a versatile and clinically validated sub-8-nm fluorescent (e.g., Cy5 dyeencapsulating) core-shell silica nanoparticle, Cornell prime dots (C’ dots), was utilized. The nanoparticle conjugate is under active investigation in Phase 1 / 2 therapeutic and image-guided surgical trials, including malignant brain tumors. Surface-adapted with short (polyethylene glycol) (PEG) ligands, a variety of targeting moieties, cytotoxic drugs, and radiolabels have been attached for targeted multimodal imaging and treatment of primary and metastatic tumors. The small size of the targeted particle platform and its well-controlled surface chemistry has led to favorable pharmacokinetic (PK) and safety profiles, reduced off-target (e.g., liver) uptake, and “target-or-clear” properties (i.e., high tumor uptake or renal clearance), which is essential for achieving efficacious outcomes over non- targeted controls.

[0116] In some embodiments, the described nanoparticle conjugates comprise ultrasmall nanoparticles (e.g., “C or C’ dots”), which are fluorescent, organo-silica core shell particles that have diameters controllable down to the sub-10 nm range with a range of modular functionalities. C or C’ dots are described by U.S. Patent No. 8298677 B2 “Fluorescent silica-based nanoparticles”, U.S. Publication No. 2013 / 0039848 A1 “Fluorescent silica-based nanoparticles”, and U.S. Publication No. US 2014 / 0248210 A1 “Multimodal silica-based nanoparticles”, the contents of which are incorporated herein by reference in their entireties. Incorporated into the silica matrix of the core are near-infrared dye molecules, such as Cy5.5, which provides its distinct optical properties. Surrounding the core is a - 38 - 12799878v1Attorney Docket No. 2018488-0014 layer or shell of silica. The silica surface is covalently modified with silyl-polyethylene glycol (PEG) groups to enhance stability in aqueous and biologically relevant conditions. These particles have been evaluated in vivo and exhibit excellent clearance properties owing largely to their size and inert surface. Among the additional functionalities incorporated into C or C’ dots are chemical sensing, non-optical (PET) image contrast and in vitro / in vivo targeting capabilities, which allow their use in visualizing lymph nodes for surgical applications, and melanoma detection in cancer. C or C’ dots provide a unique platform for drug delivery due to their physical properties as well as demonstrated human in vivo characteristics. These nanoparticle conjugates are ultrasmall (e.g., sub-8 nm) and benefit from targeted delivery (e.g., TLR-9) and EPR effects in tumor microenvironments, while retaining favorable bulk renal clearance and pharmacokinetic properties. To this end, In some embodiments, drug constructs are covalently attached to C dots (or other nanoparticles). C dot-based nanoparticle conjugates for drug delivery provide good biostability, minimize premature drug release, and exhibit controlled release of the bioactive compound. In some embodiments, peptide-based linkers are used for the described nanoparticle conjugates and other applications described herein. These linkers, in the context of antibodies and polymers, are stable both in vitro and in vivo, with highly predictable release kinetics that rely on enzyme catalyzed hydrolysis by lysosomal proteases. For example, cathepsin B, a highly expressed protease in lysosomes, can be utilized to facilitate drug release from macromolecules. By incorporating a short, protease sensitive peptide between the macromolecular backbone and the drug molecule, controlled release of the drug can be obtained in the presence of the enzyme. Interestingly, the described nanoparticles themselves exhibit intrinsic therapeutic capabilities that (1) modulate the tumor microenvironment (TME) toward a pro-inflammatory phenotype, (2) increase immune cell activation and cytotoxicity in the TME, and (3) target cancer cells directly for cell death through mechanisms, such as ferroptosis.

[0117] In some embodiments, nanoparticle conjugates comprise an ultrasmall (e.g., sub-50 nm diameter, e.g., sub-20 nm diameter, e.g., sub-15 nm diameter, e.g., sub-10 nm diameter, e.g., sub-8 nm diameter) silica nanoparticle containing a deep red / near-infrared dye (e.g., Cy5; absorption peak: 650 nm) that is covalently encapsulated within the silica-matrix. In this embodiment, due to the encapsulation of the dye and the specific design on the nanoparticle conjugates, the brightness is - 39 - 12799878v1Attorney Docket No. 2018488-0014 dramatically improved (e.g., at least 2-times, e.g., at least 10-times, e.g., at least 50-times, e.g., at least 100-times, e.g., at least 600-times) as compared to the free dye.

[0118] Nanoparticle conjugates may comprise one or more polymers, e.g., one or more polymers that have been approved for use in humans by the U.S. Food and Drug Administration (FDA) under 21 C.F.R. § 177.2600, including, but not limited to, polyesters (e.g., polylactic acid, poly(lactic- co-glycolic acid), polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2-one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; polycyanoacrylates; copolymers of PEG and poly(ethylene oxide) (PEO). In some embodiments, the diameter of the nanoparticle conjugates is not substantially increased by the one or more polymers.

[0119] The nanoparticle conjugates may comprise one or more degradable polymers, for example, certain polyesters, polyanhydrides, polyorthoesters, polyphosphazenes, polyphosphoesters, certain polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, poly(amino acids), polyacetals, polyethers, biodegradable polycyanoacrylates, biodegradable polyurethanes and polysaccharides. For example, specific biodegradable polymers that may be used include but are not limited to polylysine, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), poly(lactide-co-caprolactone) (PLC), and poly(glycolide-co- caprolactone) (PGC). Another exemplary degradable polymer is poly (beta-amino esters), which may be suitable for use in accordance with the present application.

[0120] In some embodiments, a nanoparticle conjugate can have or be modified to have one or more functional groups. Such functional groups (within or on the surface of a nanoparticle) can be used for association with any agents (e.g., detectable entities, targeting entities, therapeutic entities, or PEG). In addition to changing the surface charge by introducing or modifying surface functionality, the introduction of different functional groups allows the conjugation of linkers (e.g., (cleavable or (bio-)degradable) polymers such as, but not limited to, polyethylene glycol, polypropylene glycol, PLGA, etc.), targeting / homing agents, and / or combinations thereof.

[0121] Moreover, a label for imaging and / or radiotherapy can be attached to the nanoparticle as described herein. In some embodiments, the nanoparticle conjugate comprises a therapeutic agent, e.g., - 40 - 12799878v1Attorney Docket No. 2018488-0014 a drug moiety (e.g., a chemotherapy drug) and / or a therapeutic radioisotope. As used herein, "therapeutic agent" refers to any agent that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect, when administered to a subject.

[0122] In some embodiments, the nanoparticle conjugates described herein demonstrate enhanced penetration of tumor tissue and diffusion within the tumor interstitium, e.g., for treatment of cancer, as described in International Patent Application No. PCT / US17 / 30056 (“Compositions and Methods for Targeted Particle Penetration, Distribution, and Response in Malignant Brain Tumors,” filed April 28, 2016) by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety.

[0123] In some embodiments, the nanoparticle conjugates described herein can be used to induce ferroptosis, as described in International Patent Application No. PCT / US16 / 34351 (“Methods of Treatment Using Ultrasmall Nanoparticles to Induce Cell Death of Cancer Cells via Ferroptosis,” filed on May 26, 2016) by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety. In some embodiments, the nanoparticle conjugates described herein can be used to induce ferroptosis, as described in International Patent Application No. PCT / US18 / 63751 (“Methods of Cancer Treatment via Regulated Ferroptosis,” filed on December 4, 2018) by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety. In some embodiments, the nanoparticle conjugates described herein can be used to activate tumor cells and / or innate and adaptive immune responses within the tumor microenvironment, as well as induce immune-related necrosis among other cell death processes, as described in International Patent Application No. PCT / US19 / 66944 (“Inducing Favorable Effects on Tumor Microenvironment via Administration of Nanoparticle Compositions,” filed on December 17, 2019, by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety. In some embodiments, the nanoparticle conjugates described herein can be used to activate the tumor microenvironment, as described in International Application No. PCT / US22 / 34224 (“Nanoparticle-mediated Enhancement of Immunotherapy to Promote Ferroptosis-induced Cytotoxicity and Antitumor Immune Responses,” filed on June 21, 2022) by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety. See also, e.g., International Patent Application No. PCT / US16 / 26434 (“Nanoparticle Immunoconjugates,” filed on April 7, 2016, by Bradbury et al., the contents of which is hereby incorporated by reference in its - 41 - 12799878v1Attorney Docket No. 2018488-0014 entirety. See also, e.g., International Patent Application No. PCT / US18 / 33098 (“Ultrasmall Nanoparticles Labeled with Zirconium-89 and Methods Thereof,” filed on May 17, 2018, by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety.

[0124] In some embodiments, the nanoparticle conjugates described herein are used in combination with immunotherapy described in U.S. Provisional Application No. 63 / 532,203 (“Engineered Particle-Based Immunomodulators and Immunotherapies, and Uses Thereof.” Filed on August 11, 2023, by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety.

[0125] Moreover, use of radiolabels and / or fluorescent markers attached to (or incorporated in or on, or otherwise associated with) the nanoparticles provide quantitative assessment of nanoparticle conjugates uptake at the target site and within the body, as well as permit monitoring of treatment response. In various embodiments, modular linkers are described for incorporating targeting ligands to develop a drug delivery system with controlled pharmacological properties. The described platforms determine the influence of targeting on conjugates penetration and accumulation, thereby establishing an adaptable platform for improved delivery of a range of tractable SMIs, for example, to primary and metastatic brain tumors.

[0126] In some embodiments, a screen of different nanoparticle conjugates (e.g., AAINCs, e.g., different anti-VEGF-C' dots, e.g., DARPin-based nanoparticle conjugates) alone and / or with subtherapeutic radiation therapy (low-dose RT) can be performed to determine the degree of suppression of neuroinflammation and / or edema that promotes the growth of breast cancer brain metastases (BCBMs). Moreover, quantification of edema volumes, tumor size, and other MRI functional parameters using multiparametric MRI is envisioned. In some embodiments, the nanoparticle conjugates may be associated with MRI labels and / or optical probes.

[0127] In some embodiments, a nanoparticle conjugate is DARPin-based nanoparticle conjugate. In some embodiments, a nanoparticle conjugate is an anti-angiogenic and / or immunomodulatory nanoparticle conjugate (“AAINC”).

[0128] In some embodiments, a nanoparticle conjugate comprises one or more fluorophores. Fluorophores comprise fluorochromes, fluorochrome quencher molecules, any organic or inorganic - 42 - 12799878v1Attorney Docket No. 2018488-0014 dyes, metal chelates, or any fluorescent enzyme substrates, including protease activatable enzyme substrates. In some embodiments, fluorophores comprise long chain carbophilic cyanines. In other embodiments, fluorophores comprise DiI, DiR, DiD, and the like. Fluorochromes comprise far red, and near infrared fluorochromes (NIRF). Fluorochromes include but are not limited to a carbocyanine and indocyanine fluorochromes. In some embodiments, imaging agents comprise commercially available fluorochromes including, but not limited to methylene blue, Cy5.5, Cy5 and Cy7 (GE Healthcare); AlexaFlour660, AlexaFlour680, AlexaFluor750, and AlexaFluor790 (Invitrogen); VivoTag680, VivoTag-S680, and VivoTag-S750 (VisEn Medical); Dy677, Dy682, Dy752 and Dy780 (Dyomics); DyLight547, DyLight647 (Pierce); HiLyte Fluor 647, HiLyte Fluor 680, and HiLyte Fluor 750 (AnaSpec); IRDye 800CW, IRDye 800RS, and IRDye 700DX (Li-Cor); methylene blue; and ADS780WS, ADS830WS, and ADS832WS (American Dye Source) and Kodak X-SIGHT 650, Kodak X-SIGHT 691, Kodak X-SIGHT 751 (Carestream Health). In some embodiments, a multi-wavelength camera as described by Bradbury et al. US Publication No. US 2015 / 0182118 A1, “Systems, Methods, and Apparatus for Multichannel Imaging of Fluorescent Sources in Real Time”, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the imaging system used to image the lesion provides both static and functional assessments of the area of treatment (and its surroundings).

[0129] The surface chemistry, uniformity of coating (where there is a coating), surface charge, composition, concentration, frequency of administration, shape, and / or size of the nanoparticle conjugates can be adjusted to produce a desired therapeutic effect.

[0130] In some embodiments, the nanoprobes comprises a chelator, for example, 1,4,8,1 l- tetraazabicyclo[6.6.2]hexadecane-4,l 1- diyl)diacetic acid (CB-TE2A); desferoxamine (DFO); diethylenetriaminepentaacetic acid (DTPA); 1,4,7, 10-tetraazacyclotetradecane- 1,4,7, 10-tetraacetic acid (DOTA); thylenediaminetetraacetic acid (EDTA); ethylene glycolbis(2-aminoethyl)-N,N,N',N'- tetraacetic acid (EGTA); 1,4,8,1 l-tetraazacyclotetradecane-l,4,8,l l-tetraacetic acid (TETA); ethylenebis-(2-4 hydroxy-phenylglycine) (EHPG); 5-Cl-EHPG; 5Br-EHPG; 5- Me-EHPG; 5t-Bu- EHPG; 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA); dibenzo-DTPA; phenyl-DTPA, diphenyl-DTPA; benzyl-DTPA; dibenzyl DTPA; bis-2 (hydroxybenzyl)-ethylene- diaminediacetic acid (HBED) and derivatives thereof; Ac-DOTA; benzo-DOTA; dibenzo-DOTA; - 43 - 12799878v1Attorney Docket No. 2018488-0014 1,4,7-triazacyclononane Ν,Ν',Ν"- triacetic acid (NOTA); benzo-NOTA; benzo-TETA, benzo-DOTMA, where DOTMA is 1,4,7, 10-tetraazacyclotetradecane-l,4,7,10-tetra(methyl tetraacetic acid), benzo- TETMA, where TETMA is 1,4,8,1 l-tetraazacyclotetradecane-l,4,8,l l-(methyl tetraacetic acid); derivatives of 1,3-propylenediaminetetraacetic acid (PDTA); triethylenetetraaminehexaacetic acid (TTHA); derivatives of l,5,10-N,N',N"-tris(2,3- dihydroxybenzoyl)-tricatecholate (LICAM); and l,3,5- N,N',N"-tris(2,3- dihydroxybenzoyl)aminomethylbenzene (MECAM), or other metal chelators.

[0131] In some embodiments, a nanoparticle conjugate comprises an azide moiety. In some embodiments, an azide moiety is attached to an antibody fragment for conjugation to a nanoparticle described herein. In some embodiments, azide moieties are attached to one or more antibody fragments (e.g., total of a single member or total of different members) (e.g., anti-VEGF, e.g., anti- VEGF-A, anti-VEGF-B, or a combination thereof), for instance, for conjugation to a nanoparticle described herein.

[0132] In some embodiments, the nanoconjugate comprises more than one chelator. In some embodiments the radioisotope-chelator pair is89Zr-DFO. In some embodiments the radioisotope-chelator pair is177Lu-DOTA. In some embodiments, the radioisotope-chelator pair is225Ac-DOTA. EXEMPLIFICATION

[0133] In order that the application may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting in any manner. EXAMPLE 1 – Developing and characterizing ultrasmall VEGF-targeting nanoparticle conjugates

[0134] The present example describes an exemplary development and evaluation of an efficacious ultrasmall VEGF-targeting particle adjuvant that can suppress inflammation-mediated processes / edema and growth in BCBM models – serving as a safe alternative to corticosteroids. Towards this end, a versatile and clinically validated sub-8-nm fluorescent (e.g., Cy5 dyeencapsulating) core-shell silica nanoparticle, Cornell prime dots (C’ dots), is utilized. The nanoparticle conjugate is under active investigation in Phase 1 / 2 therapeutic and image-guided surgical trials, including - 44 - 12799878v1Attorney Docket No. 2018488-0014 malignant brain tumors. Surface-adapted with short (polyethylene glycol) (PEG) ligands, a variety of targeting moieties, cytotoxic drugs, and radiolabels have been attached for targeted multimodal imaging and treatment of primary and metastatic tumors. The small size of the targeted particle platform and its well-controlled surface chemistry has led to favorable pharmacokinetic (PK) and safety profiles, reduced off-target (e.g., liver) uptake, and “target-or-clear” properties (i.e., high tumor uptake or renal clearance), which is essential for achieving efficacious outcomes over non-targeted controls. The process is summarized in FIG. 1. The statistically significant growth inhibition and prolonged survival for malignant brain tumors were found in both platelet-derived growth factor (PDGF)-B-driven high-grade glioma (RCAS / tv-a glioma) and epidermal growth factor (EGFRvIII) patient-derived xenograft (PDX) models using doxorubicin-conjugated C’ dots. MEASUREMENTS

[0135] Candidates are evaluated and compared at each stage of technology development based on key selection criteria and discrete measurements – illustrated as a series of screening steps (boxes) in FIG. 1. Through these measurements, potent, non-toxic VEGF-targeting C’ dots (or VEGF-C’ dots) are developed and identified (SA1 in FIG. 1) whose combined antiangiogenic and immune modulatory properties can be leveraged to not only limit inflammatory changes associated with tumor growth and RN, but serve as a potentially synergistic partner with subtherapeutic (15-20 Gy) RT to limit suppressive activities and prolong survival over controls (SA2 in FIG. 1). Treatment responses reflect the contributions of RT and the particle adjuvant. The brain-tropic metastatic model systems used herein aim to better recapitulate disease progression in patients and will allow mechanisms driving inflammation within the TME to be probed. Once measurements in SA2 in FIG. 1 are met and a VEGF-C’ dot identified, translational and safety studies are conducted.

[0136] The following examples support that the development of a first-in-kind ultrasmall VEGF-targeting C’ dot adjuvant-therapeutic can mitigate both inflammatory and immune suppressive activities driving BCBM growth and the onset / progression of RN following single-fraction RT, in turn, conferring a survival advantage over controls. - 45 - 12799878v1Attorney Docket No. 2018488-0014 EXAMPLE 1A - Identify tunable structural properties of anti-VEGF scFv conjugated C’ dot adjuvants that maximally enhance immunogenicity, immune / stromal cell function, and cytotoxic responses in BC cells. 1A.a. Develop and characterize anti-VEGF scFvs, followed by conjugation of one or more individual VEGF ligands (VEGF-A, VEGF-B, or VEGF-A and -B) to C’ dots to maximize anti-inflammatory properties.

[0137] Although the base particle possesses intrinsic therapeutic properties, modification of specific structural parameters, such as the surface attachment of anti-VEGF scFvs, can augment the anti-inflammatory properties of the particle and other biological activities. The surface of C’ dots can be modified with variable numbers of synthetically engineered and functionally distinct vascular endothelial growth factor (VEGF) family of proteins (scFvs) – either singly or in combination – to enhance their structural and anti-inflammatory properties. This includes (i) modulating EC function, (ii) polarizing macrophages towards a pro-inflammatory phenotype, and (iii) increasing immunogenicity of brain-tropic TNBC cells derived from brain metastatic TNBC models. Generation and characterization of anti-VEGF-A / -VEGF-B scFvs.

[0138] Given that human and murine VEGFs are 85% identical in sequence, amino acid sequences corresponding to overlapping variable light and heavy regions of both murine and human anti-VEGF-A and anti-VEGF-B antibodies are used. Genetic sequences (gBlocks) are constructed using a codon improvement tool (Integrated DNA Technologies, Newark, NJ). These incorporate signal peptide sequences for secretion, His-tags for purification, and a “free” cysteine at the C-terminus for attaching scFvs adapted with azides. Linearized gBlocks are cloned into lentiviral vectors for transduction of Lenti-X cells (TakaraBio) and synthesis / secretion of scFvs. Vectors co-express a reporter protein for assessing transduction efficiency and enriching positively transduced cell populations. After sorting, cells can be cultured, and supernatant processed to obtain purified His- tagged proteins. To characterize the size, sequence, specificity and stability of these scFv constructs, the followings can be performed, respectively: western blot, mass spectroscopy, competitive binding assays (i.e., using anti-VEGF-A / VEGF-B antibodies), and size-exclusion chromatography. Synthesis of VEGF scFv-C’ dots with varying ligand density and non-targeted controls (C’ dots). - 46 - 12799878v1Attorney Docket No. 2018488-0014

[0139] VEGF-targeting particles, referred to as VEGF-C’ dots, and non-targeted C’ dot controls, as well as deferoxamine- (DFO-) chelator modified C’ dots, can be synthesized, purified, and characterized following well established synthesis methods and advanced characterization techniques. Characterization utilize high-performance liquid chromatography (HPLC) methods, pioneered by the team and facilitating the clinical translation of final C’ dot products. DFO and VEGF conjugation to C’ dots are accomplished by the post-PEGylation surface modification by insertion (PPSMI) method employing validated protocols from earlier scFv ligand studies. Small functional aminopropyl- trimethoxy-silane (APTMS) is inserted between PEGs on the silica surface for further reactions with functional moieties. Resulting NH2-C’ dots are first reacted with 1-(4-isothiocyanatophenyl) containing p-SCN-Bn-DFO. Dibenzocyclooctyne (DBCO) containing DBCO-PEG4- Nhydroxysuccinimidylester (DBCO-PEG4-NHS ester) is then added, yielding DBCO-DFO-C’dots. Azide containing VEGF scFv’s can then efficiently be “clicked” onto the particles via azide-alkyne cycloaddition (in three batches: either single VEGF-A or -B, or combined VEGF-A / B). The number of DFO chelators, DBCOs, and scFv’s are controlled by the reaction concentration ratios of ligands to particles (aiming at 1-2 DFO’s, 20-30 DBCO’s, and 1-3 scFv’s per particle), with particle concentrations derived from fluorescence correlation spectroscopy (FCS). DFO’s / DBCO’s / VEGF’s per particle is quantified by UV-Vis spectral deconvolution methods driven by Cy5 particle fluorescence and FCS-derived particle concentrations as described elsewhere.

[0140] Structural measurements used as go / no-go screening criteria: (i) scFv, C’ dot purity greater than 95%; (ii) C’ dot hydrodynamic diameter less than 8 nm; (iii) number of Cy5 dyes (greater than 1); (iv) VEGF scFvs per C’ dot: 1– 3; (v) DBCO ligands per C’ dot: 20 – 30; (vi) DFO ligands per C’ dot: 2 – 5. 1.A.b. Assess differential modulatory effects of VEGF-targeting C’ dots developed in this Example, over non-targeting C’ dots, on endothelial cells (ECs), myeloid cells, and tumor cells to identify candidates for in vivo studies.

[0141] In vitro cell screening assays are conducted using both murine and human BC cell lines, ECs, and bone marrow derived macrophages (BMDMs) exposed to non-targeting and VEGF-targeting particles in order to identify candidates that enhance anti-inflammatory and cytotoxic responses. These - 47 - 12799878v1Attorney Docket No. 2018488-0014 studies serve to inform in vivo work, as well as yield mechanistic insights regarding distinct particle features driving anti-inflammatory and / or cell death responses. All studies are performed in triplicate. Breast Cancer Cell lines.

[0142] Luciferase-expressing (luc+) murine brain-tropic TNBC cell lines (4T1-, E0771- BrM2) were a kind gift from Dr. A. Boire (Memorial Sloan Kettering, NY). The human brain-tropic BC cell line, MDA-MB-231-BrT2, or BrT2), were supplied by Dr. D. Lyden. Preparation of BMDMs and ECs.

[0143] BMDMs are prepared from femurs and tibias of tumor-bearing BALB / C mice. After lysis of red blood cells, the remaining cells are plated in bone macrophage media (DMEM, 20% FBS plus penicillin / streptomycin, and 50 ng / ml M-CSF) and incubated for 6 days. Both murine (C166, #CRL-2581) and human (human umbilical vein endothelial cells) ECs, from ATCC, are cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) and Endothelial Cell Growth Media, respectively. Competitive enzyme-linked immunosorbent assay (ELISA).

[0144] VEGF constructs are designed to bind to soluble VEGF, similar to the mechanism utilized by bevacizumab. Using bevacizumab as an internal reference, the IC50 of both VEGF-C’ dot constructs and VEGF scFvs are determined over a range of concentrations using an ELISA assay. Binding Affinity.

[0145] Concentration- (100nM–15μM) and time- (4–72 hours) dependent binding of VEGF-C’ dots bearing different numbers of scFvs, as against non-targeting C’ dots, are evaluated using supernatants derived from GFP+ / luc+ 4T1- BrM2, E0771-BrM2, and BrT2 cell populations and a competitive ELISA assay. Expression profiling.

[0146] Using VEGF-C’ dot constructs, upregulation of gene expression signatures related to iron metabolism (e.g., Fth1, Tf, Slc40a1), ferroptosis (e.g., Slc7a11 / Slc3a2 cystine / glutamate antiporter), antigen presentation (e.g., H2-D1, H2-T23, Tap1, Tap2) and angiogenesis (e.g., CD31, VEGF, Tie2, P-selectin) in tumor cells, are assessed. Changes in gene expression are measured using - 48 - 12799878v1Attorney Docket No. 2018488-0014 quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) with TaqMan assays in duplexed reactions with controls (e.g., Gapdh). Results are analyzed as a fold-change using the DDCT method93 with log2- transformation. Significantly upregulated gene expression signatures can be validated by flow cytometry. Once optimal cell-specific conditions are established, bulk RNA-seq experiments will also be performed to further expand upon classes of genes modulated by different VEGF-C’ dot compositions and ligand constructs. Cytokine release assays.

[0147] Changes in gene expression profiles related to immunostimulatory or immunosuppressive cytokines from I.b.5 are validated using targeted cytokine proteome assays. Samples are quantified using a concentration standard curve, then normalized based on reference samples containing known protein or pro-inflammatory cytokine / chemokine concentrations. Cell death assays.

[0148] Cell death induction by VEGF-C’ dot constructs are assessed by incubating cells over a range of particle concentrations (0–15 μM), and cell viability assayed up to 72 hours using live cell imaging with the cell death indicator Sytox Red. 1.A.c. Screen the temporal evolution of brain metastases for each model following intracardiac injection using serial bioluminescence imaging (BLI) and MR imaging to select early and late times for particle treatment. Animal Models and Tumor Inoculation.

[0149] All experiments are performed in accordance with protocols approved by the WCM Institutional Animal Care and Use Committee. Using ultrasound guidance, 1x104 luc+4T1-BrM2 / E0771-BrM2 cells or BrT2 cells are injected into the left ventricle of 6-8-week-old BALB / C or BALB / C nu / nu mice, respectively (♀, Jackson Laboratories, ME) to generate BCBMs. Serial BLI monitoring of tumor burden.

[0150] All luc+ BCBM mice using BLI (n=494 mice) are monitored as early as 5 days post- intracardiac injection, before macroscopic lesions are noted. Given the short lifespan of BCBM (~3-4 weeks), disease progression is monitored biweekly with BLI or until adverse effects are observed (e.g., - 49 - 12799878v1Attorney Docket No. 2018488-0014 greater than 20% weight loss, lethargy). At the time of initial BLI signal detection, T2-weighted MRI is also acquired to confirm the presence of inflammatory changes / tumor. Based on multimodal imaging findings, an early and late time point are selected for initiating treatment studies (EXAMPLE 3B). Longitudinal screening and multiparametric MRI.

[0151] Metastatic growth and inflammatory changes / edema are monitored in a 7T Bruker magnet over a ~3-4-week period in representative numbers of BCBM mice across models (n=84) using high-resolution T2-Weighted and T2-Fluid Attenuation Inversion Recovery (FLAIR) MR imaging sequences (TR:10 s; TE:48 ms; TI:2 s) MRI. T2-mapping is performed using a 12-echo sequence and exponential fitting for reproducibly quantitating T2-relaxation times. T2 histogram analysis will then yield accurate volumetric measurements of edematous tissue (mm3) using (mean+ / - standard deviation, or μ±2s) cutoffs in the region. Multiparametric MRI is also performed in a representative number of BCBM animals (n=75) pre- and post-treatment, and the following functional parameters quantitated for comparison across treatment cohorts: (i) apparent diffusion coefficients (ADCs) using diffusion- weighted imaging (DWI); these may increase in regions of RN, indicative of lysed cell membranes following RT and (ii) relative cerebral blood volumes (rCBV) (angiogenesis) and BBB permeability surface area products (Ktrans) using dynamic contrast enhanced MRI (DCE-MRI) following i.v.- injection of gadolinium (Gadovist).

[0152] The key in vitro biological and imaging measurement is the selection of VEGF C’ dot probes for in vivo PK studies per the following go / no-go criteria: (i) Increase in IC50: VEGF scFv-C’ dots greater than IC50 (scFv); (ii) specificity (block) greater than 50%.

[0153] It is described that VEGF scFvs can be conjugated to C’ dots using a range of scFv numbers per established protocols. High purity and affinity can be obtained for the VEGF scFv constructs generated (see FIG. 1). - 50 - 12799878v1Attorney Docket No. 2018488-0014 EXAMPLE 1B - Using VEGF-C’ dots alone or with RT evaluate time-dependent changes in inflammatory and cytotoxic responses, growth inhibition, and survival in BCBM models. 1.B.a. Conduct screening PK studies of zirconium-89 (89Zr)-labeled VEGF-C’ dot candidates in BCBM models.

[0154] Establishing the safety and efficacy of investigational agents requires a detailed understanding of their PK, among other properties. Based on structural and biological improvements (see EXAMPLE 3A), two VEGF-C’ dot candidates are conjugated with DFO and radiolabeled with zirconium-89 (89Zr) according to well-established protocols and measurements specified in FIG. 1, in order to assess the influence of particle surface chemistry on PK and its targeted delivery to both murine and human BCBM models.

[0155] Screening PK / BD (n=5 mice / C’ dot tracer; ~20 μCi / mouse) and serial positron emission tomography (PET) imaging (n=5 mice / C’ dot tracer; ~300 μCi / mouse) studies are performed at 4, 24, and 72 hr after i.v.-injection of two 89Zr-DFO-VEGF-C’ dots in both (i) non-tumor-bearing mice (n=5 / cohort; ♀) and (ii) BCBM mice (n=5 / cohort; ♀) per the well-established protocols presented herein. Measurement endpoints are described in FIG. 1.

[0156] The key biological / radiological measurements is that candidate C’ dot probes are selected for therapeutic studies using go / no-go criteria: (i) specific activity (particle tracer) greater than 1 x 104 Ci / mol and % max tumor uptake greater than 10 %ID / g. 1.B.b. Investigate contributions of particle-driven anti-tumor responses needed to maximize tumor regression and inflammatory changes within the TME at pre-selected early and late time points using BLI and MRI, with and without a single-fraction dose (15-Gy) of irradiation, focusing on optimal sequencing of treatments.

[0157] Advancing new non-steroidal treatment regimens that can suppress inflammation- mediated processes driving disease and RN in order to control tumor growth and treatment outcomes would represent a significant paradigm shift. Using both murine and human BCBMs, a VEGF-C’ dot candidate is i.v.-injected, at both early and late time points to assess differential treatment responses relative to those of non-targeting C’ dots using BLI and multiparametric MRI. The VEGF-C’ dot - 51 - 12799878v1Attorney Docket No. 2018488-0014 candidate that significantly minimizes inflammation, reduces tumor growth, and improves survival is then used to (i) develop new synergistic combinatorial paradigms with subtherapeutic (s.t.) RT to augment outcomes in the same models, as well as (ii) investigate inflammatory changes / edema induced in the setting of RN, relative to controls, in a murine BCBM model following high-dose RT and different particle dose schedules. Particle-driven anti-tumor responses in BCBM models.

[0158] Briefly, using methods and particle concentrations established herein select murine BCBMs (♀, 6-8 weeks old, n=10 mice / cohort) are treated at ~7 days after intracardiac injection with an i.v.-injected multidose regimen (i.e., n=3 doses every 3 days, or Q3Dx3) of one (or more) VEGF-C’ dot candidates, non-targeting C’ dots, or saline vehicle. Growth inhibition can be monitored (n=3 cohorts) with BLI biweekly and multiparametric MRI weekly (1.c.2, 1.c.3) over a ~3-4-week period, and a survival study performed to assess survival benefit (n=10 mice / cohort). At study termination, brain tumors are harvested per established protocols94 for H&E staining and immunophenotyping (IP). Treatment Planning and Irradiations.

[0159] Using X-Rad Small-Animal Radiation Therapy (SmART)+ Biological Irradiator with the appropriately sized collimator (lesion size-dependent) and its SmART Advanced Treatment Planning system, a treatment plan for each mouse is derived in order to deliver the prescribed absorbed dose uniformly to the full depth of the brain metastasis and contoured to the metastasis. Different combinations of the number of beams and their angulation and relative weights (beam-on times) are evaluated using the treatment planning software and the optimum treatment plan selected and applied. For the treatment plan selected, isodose contours in and around the lesion and the lesion dose-volume histogram are provided. Combination strategies with single-fraction 15-Gy RT to reduce inflammation and augment efficacy.

[0160] FIG. 2 describes n=7 treatment arms (G1-7; n=15 mice / cohort): RT+ particles (n=2) and controls (G1, G2, G4-6). Using the same models described herein, as well as the RT treatment plan established herein, single-fraction (15-Gy), RT are combined with the first dose of VEGF-C’ dots at 7 days post intracardiac implantation of cells (Option 1). Alternatively, RT can be administered 14 days later (Option 2). This design will allow for determining whether the order of administration of - 52 - 12799878v1Attorney Docket No. 2018488-0014 these agents is important. For ‘early’ or ‘late’ RT, particles are injected in triplicate on days 0, 3, and 6. Imaging procedures are repeated for monitoring growth inhibition, and then animals and harvest brain tumor and serum specimens (n=3 / cohort) are euthanized at the study measurements (murine model only) to perform cytokine analysis, IP, and single-cell RNA-sequencing (scRNA-seq). Additional brain tumor specimens (n=3 / cohort) are employed to quantitatively analyze for the presence and distribution of VEGF-C’ dots using super-resolution microscopy techniques described earlier. Animals are euthanized for signs of distress, lethargy, and body weight changes (greater than 20%). Urinary specimens are then collected to analyze VEGF-C’ dot size variations by FCS before / after treatments using protocols described elsewhere. Toxicology studies are also performed posttreatment (Option 1 only) on representative mice from each murine cohort (n=3) for analyzing tumors, serum cytokines, and pathology of select major organs / tissues (e.g., liver, lung, blood) using H&E staining. Survival benefit.

[0161] Using the combination arm from each model described herein that resulted in significantly reduced inflammation and / or maximum growth inhibition, along with the same control arms, the same procedures is repeated to assess survival benefit for both models. Combination strategies with single-fraction 50-Gy RT to induce RN.

[0162] FIG. 3 describes n=8 treatment arms (G1-8; n=15 mice / cohort): RT+ particles (n=3) and controls (G1, G5-8). Using the same murine model as described herein and following the RT treatment plan established herein, single-fraction (50-Gy), we will administer RT at the same time point for all treatments (i.e., 14 days post-intracardiac implantation). The three-treatment arms represent differences in the time of initiation of i.v.-injected VEGF-C’ dots, permitting the best sequence strategy to be determined for abating inflammatory changes and maximizing efficacy. In this paradigm, n=4 particle doses, i.e., Q3Dx4, that ‘straddle’ the RT dose, are being used: treatment #1 (3 particle doses before / 1 dose after), treatment #2 (2 particle doses before / 2 doses after), and treatment #3 (1 particle dose before / 3 doses after). As described herein, imaging procedures are repeated for monitoring growth inhibition, and then animals and harvest brain tumor specimens are euthanized (n=3 / cohort) at the study measurements for performing only IF to assess alterations in markers reflecting RN, relative - 53 - 12799878v1Attorney Docket No. 2018488-0014 to control cohorts, including caspase-3, gH2AX, VEGF, IL-6, TNF-a. Animals are euthanized for signs of distress, lethargy, and body weight changes (greater than 20%).

[0163] The key in vivo biological measurement is that the VEGF-C’ dot probes are selected using go / no-go criteria: (i) Survival benefit: combo RT+VEGF-C’ dot greater than RT, VEGF-C’ dot alone, RT+DEX; (ii) Decrease in Inflammation: Decrease in Edema volume, Decrease in ADC (MRI): VEGF-C’ dot+RT greater than RT, RT+DEX, (iii) Increase in T cell activity (CD8+ / Treg), and (iv) reverse / limit immune suppression (Decrease in MDSC, Decrease in Tregs, Decrease in PD-1); Decrease in inflammation: Decrease in VEGF, Decrease in IL-6, Decrease in TNF-a. 1.B.c. Determine the optimal timing and dosing regimen for particle administration needed to mitigate the onset and progression of RN following a single-fraction, 50-Gy dose of hemispheric irradiation and MRI. Statistical analysis.

[0164] For in vitro assays described herein, continuous measurements are analyzed by reporting summary statistics and 95% confidence intervals, while binomial proportions (i.e., gene expression fold-change greater than 2) can be reported with 95% Clopper-Pearson intervals. Bulk sequencing data is aligned to reference genome and transcripts quantified using RSEM. Data is normalized using DEseq2 and differential expression analysis done to identify gene sets that are altered; this can be followed by gene set enrichment and pathway analyses. Briefly, as described herein, animal studies with longitudinal outcomes (i.e., PK, etc) are reported per time point, and treatment groups are compared using a rank-based test. Point estimates and confidence intervals for gene expression changes (reported as log-transforms) are back-transformed and reported as foldchange using the DDCT method. As described herein, an ANOVA is performed for comparison of treatment response parameters (tumor sizes, edema volumes and MRI biomarkers) in animal studies using group differences as factors, and then separately with each parameter as the outcome. Significant group differences are then followed up with post-hoc tests for pairwise group comparisons between one / combination of groups using Scheffé Test. Mouse survival is estimated separately within each group using Kaplan-Meier methods and summarized for inter-group comparisons using hazard ratios and 95% confidence intervals and compared with a log-rank test. With n=10 mice per cohort, there can - 54 - 12799878v1Attorney Docket No. 2018488-0014 be more than 80% power to detect a four-fold difference (HR=4.0) between two cohorts. For scRNA studies, the Bioconductor workflow is followed for pre-processing and clustering and lineage inference is performed. Pathway and gene set overdispersion analysis (PAGODA) can be used to detect transcriptional heterogeneity within a cell population, and a weighted principal component analysis (PCA) is conducted for each gene set defining immune / tumor cell populations and states. EXAMPLE 2 - Developing high-affinity and species-specific MUC16 and MSLN DARPin-based binders and assess binding kinetics, specificity, and stability.

[0165] The present example shows compositions and methods of generating MUC16 and MSLN DARPin-based binders to develop DARPin-C’ dots for enhancing MUC16-targeting MSLN- CAR T cell cytotoxicity and OC immunogenicity. DARPin-based binders that selectively recognize membrane-bound domains of MUC16 and MSLN are developed, along with controls, and DARPin candidates conjugated to C’ dots. Structural properties are improved to (i) yield high specificity, (ii) enhance CAR T cell cytotoxicity; (iii) increase immunogenicity of OC cells, and (iv) maximize OC cell death. For example, adapting C’ dots with high-affinity MUC16-CD (or MUC16) and MSLN Designed Ankyrin Repeat Protein (DARPin)-based binders (less than 20 kDa) designed to target human and murine MUC16 and MSLN antigens can further augment anti-tumor responses. (See FIG. 7)

[0166] This Example also describes assessment of biological properties of DARPin-based binders in MUC16ecto murine and human OC cell lines, and to select candidates for C’ dot conjugation. One (or more) MUC16- (or MSLN-) DARPin-C’ dots can then be evaluated in syngeneic and humanized models, with proinflammatory phenotypic responses, cytotoxicity, and markers of T- cell activation, exhaustion and persistence monitored after i.v.-injection to assess improvements in suppressive activities. Data can inform combinatorial strategies using a DARPin-C’ dot and MUC16- targeting MSLN-CAR T cells, with the goal of producing a clinically translatable product that limits suppression, prolongs survival, and reduces toxicity. (See FIGs. 8-12)

[0167] By attaching MSLN- and MUC16-DARPins individually to the particle surface, marked increases in OC and T cell internalization accompanied by amplified anti-tumor immune responses and targeted cell kill were observed. Additionally, it is described herein that combining a DARPin-C’dot - 55 - 12799878v1Attorney Docket No. 2018488-0014 with MSLN-CAR T cells limit suppression and improve treatment outcomes in OC models over controls.

[0168] A high-diversity DARPin library is being constructed using degenerate oligonucleotides encoding diversified binding surfaces with overlap extension PCR and modular assembly, and non- selective, as well as selective binders developed, the latter for targeting MUC16 and MSLN. High- throughput computational screening tools (e.g., AlphaFold2 / 3, Schrödinger) are used to screen this library in order to identify potential therapeutic candidates with high specificity / affinity to MUC16 and MSLN targets. Phage display screening is used to enrich target-specific DARPins through biopanning. Binding kinetics of selected high-affinity DARPins are assessed by surface plasmon resonance to determine binding affinity (KD), association (Ka), and dissociation (Kd) rates. Target engagement and specificity are confirmed by ELISA and DARPin controls. To ensure efficient and scalable production, high-affinity DARPins are cloned into a pET-28a(+) expression vector and expressed in E. coli (DE3). DARPins are adapted with N-terminal TEV-6xHis tags for affinity purification by Ni-NTA chromatography and to facilitate His-tag removal via TEV cleavage. EXAMPLE 3 - Methods of improving Immune Suppressive Activities and Therapeutic Efficacy in High-Grade Ovarian Cancer Using AI-guided Ultrasmall Targeted Particle Therapies in Combination with MUC16-targeting Mesothelin CAR T cells.

[0169] Patients with advanced and refractory ovarian carcinomas have a very poor prognosis, with an overall 5-year survival of 20%. Only 5-10% of patients are ultimately cured with standard treatments. Ovarian cancers (OC) are immunogenic tumors, highlighting their potential for treatment with immune-based therapies, such as chimeric antigen receptor (CAR) T cells, which are FDA approved for treating multiple hematologic malignancies. However, only limited efficacy has been achieved in solid tumor malignancies, including relapsed / refractory OC. CAR T cells designed to target glycoproteins overexpressed on OC cells and promoting tumor progression, such as mucin-16 (MUC16, or CA125) and mesothelin (MSLN), have generally led to modest treatment responses. This has largely been attributable to an immune suppressive tumor microenvironment (TME) that inhibits the function of tumor-specific effector cells and results in poor effector T cell persistence. Leveraging state-of-the-art combinatorial partners that effectively modulate TME suppressive activities and induce cytotoxic responses is essential for augmenting CAR T cell function, as well as for recruiting - 56 - 12799878v1Attorney Docket No. 2018488-0014 endogenous anti-tumor immune effectors targeting a broader array of tumor cell antigens and neo- antigens. Newer generation nanomedicines exhibiting intrinsic therapeutic properties offer unique opportunities to address these challenges. The present disclosure shows a clinically validated ultrasmall fluorescent (Cy5) particle, Cornell prime dots (C’ dots) without an attached cytotoxic drug, acts as an adjuvant-therapeutic through its enhancement of immunogenicity, cytotoxicity, and IT responsiveness over controls in highly suppressive solid TMEs. Using a murine ID8 OC model transduced with human (h)MUC16, non-targeting C’ dots are shown to be reversing suppressive activities and leading to a sustained survival benefit in combination with low-dose IL-12-secreting CAR T cells targeting hMUC16ecto, the retained oncogenic domain of MUC16 on cancer cells. High- affinity MSLN or hMUC16ecto Designed Ankyrin Repeat Protein (DARPin)-based binders (less than 20 kDa) are tested for surface-conjugation to C’ dots. These therapeutic conjugates are designed to target MSLN and hMUC16ecto antigens on human (OVCAR3) and hMUC16ecto-transduced murine cell lines (i.e., ID8, c-Myc+p53- / - (or MP) and c-Myc+ p53- / - BRCA1 (BReast CAncer gene 1) (or MPB1)) that, in turn, enhance anti-tumor responses over controls. Using AI-driven approaches (FIG. 33), an important goal is to identify DARPin-C’ dot candidates whose immunomodulatory properties can be leveraged to synergize with a new hMUC16ecto-targeting MSLN-CAR T cell that maximizes efficacy and enhances anti-tumor immunity in clinically relevant hMUC16 OC models, while mitigating adverse events. A rigorous, structure-based computational design pipeline is used to engineer a diverse library of DARPin scaffolds specifically tailored to bind key epitopes on both MSLN and MUC16; additional DARPin scaffolds targeting epitopes on vascular endothelial growth factor receptor (VEGFR) and human epidermal growth factor receptor 2 (HER2) have already been generated. The present AI strategy integrates advanced docking algorithms and consensus scoring to predict and rank candidate binders with high affinity and specificity ensuring robust in silico validation. Protein expression, purification, and biophysical assays are conducted to assess MSLN- and MUC16- targeting DARPin binding kinetics and stability in select OC cell lines. Selected candidates are conjugated to particles, and structural properties of DARPin-C’ dots that maximally enhance immunogenicity, immune cell function, and cytotoxicity in OC and immune cells are identified and improved relative to controls. DARPin-C’ dot candidates are radiolabeled with zirconium-89 for multimodal (i.e., positron emission tomography and optical) imaging to advance sensitive early disease detection approaches and imaging biomarkers, and to assess pharmacokinetic (PK) profiles in select - 57 - 12799878v1Attorney Docket No. 2018488-0014 OC models after systemic injection. Candidates exhibiting favorable profiles are selected for single- agent therapy studies in clinically relevant MP and MPB1 models. Alterations in immune cell activation, proinflammatory phenotypes, cytotoxicity, and markers of T-cell activation, exhaustion, and persistence are examined with the aim of improving efficacy, reversing immune suppression, and elucidating cell-specific immunomodulatory and cytotoxic mechanisms. A DARPin-C’ dot is combined with hMUC16ecto-targeting MSLN-CAR T cells to yield synergistic responses in a humanized OC model that lead to prolonged survival, improved CAR T cell effectiveness, and reductions in off-target toxicity – with an eye towards product translation. EXAMPLE 4 – Workflow for Validation of Sequences

[0170] Among other things, the present example describes a workflow for validation of DARPin sequences. A first step is to obtain target structures by checking protein data bank (PDB). If target structures were unavailable in PDB, Alphafold 2 is used to predict the structures. Next, a DARPin scaffold can be selected by retrieving a suitable scaffold form the PDB. A selected scaffold will then be docked to the target, followed by improving variable positions and generating candidate sequences to design DARPin sequences. Designed candidates are then scored based on predicted binding affinity, leading to the selection of top candidate. At this stage, the top-ranked sequence is synthesized and tested using experimental methods (e.g., lab screening). If experimental validation suggests unsatisfactory binding, the process can be iterated through design DARPin sequences. An exemplary process is illustrated in FIG. 4.

[0171] This example is moderately complex, requiring planning, execution, and review. Planning Phase includes defining the scope, setting goals, and creating a timeline, which may take a few days to 1 week. This phase involves understanding the target's requirements and aligning resources. Next is execution phase which is performing the actual work to achieve the target. This phase may take a few weeks to a few months, depending on complexity. For a moderately complex target, this typically takes 2–8 weeks. This is the most variable phase, as it depends on the nature of the work and available resources. Review and Adjustment Phase is the next step. In this phase, the results are reviewed, adjustments can be made, and the target can be finalized. This phase may take 1– 2 weeks. This phase ensures the target meets the desired outcomes and addresses any issues. Total Time Estimate per Target is as follows: Planning: ~1 week, Execution: ~2–8 weeks, Review and - 58 - 12799878v1Attorney Docket No. 2018488-0014 Adjustments: ~1–2 weeks, Total: Approximately 4–11 weeks (1–3 months). Simpler targets may take less time (e.g., 2–4 weeks), while highly complex targets could take longer (e.g., 3–6 months or more). EXAMPLE 5 - Cloning, Expression, Purification, and Characterization.

[0172] To allow for the production of a bispecific HER2×VEGF DARPin, a modular construct by fusing a high-affinity anti-HER2 DARPin G3 and an anti-VEGF DARPin Abicipar pegol was designed through a flexible (G₄S)₃ linker, with an N-terminal 6×His-tag-TEV (tobacco etch virus protease) protease cleavage site and a C-terminal single cysteine residue to facilitate downstream site- specific conjugation (FIG. 13A, FIG. 13B). Monospecific anti-HER2 and anti-VEGF DARPins were similarly cloned into pET28a(+) vectors for comparative studies (FIG. 15, Panels c–f). The bispecific and monospecific DARPins were successfully transformed into E. coli BL21(DE3) cells (FIG. 16), and recombinant expression is induced using isopropyl β-D-1-thiogalactopyranoside (IPTG). Soluble expression is achieved under standard conditions, with the majority of the DARPin proteins recovered in the supernatant following bacterial lysis and centrifugation (FIG. 16, Step 5). Yields of the bispecific DARPin are highly reproducible across independent batches, averaging approximately 100– 130 mg / L (Table 1, and FIG. 6). Table 1. Expression scale and protein yield of HER2×VEGF bispecific DARPin. Summary of recombinant HER2×VEGF bispecific DARPin protein yields (mg / L) obtained from small- (30 mL) and medium-scale (300 mL) E. coli expression batches. HER2xVEGF DARPin DARPin protein yield, mg / L Expression Scale, mL

[0173] DARPin purification is performed using nickel–nitrilotriacetic acid (Ni-NTA) affinity chromatography exploiting the designed N-terminal 6xHis-tag, followed by size-exclusion - 59 - 12799878v1Attorney Docket No. 2018488-0014 chromatography (SEC) to achieve high monomeric purity (FIG. 16, Steps 6–7). Size-exclusion chromatography (SEC) profiles show a predominant monomeric peak for the HER2×VEGF bispecific DARPin at approximately 30.1 kDa, with monospecific DARPins eluting at about ~15.9 kDa (FIG. 13C). Purified proteins were further characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), confirming molecular weights and successful removal of major contaminants (FIG. 13D). As described herein, minor dimer formation was detected among all three kinds of DARPins expressed, consistent with intermolecular disulfide bond formation. Under non- reducing conditions, the HER2×VEGF DARPin exhibited 1.7 % dimer content after SEC purification, which was further reduced to 0.2 % and zero following treatment with 1 mM tris(2- carboxyethyl)phosphine (TCEP) or β-mercaptoethanol (BME), respectively (Table 2, FIG. 13D). Table 2. Quantification of monomer and dimer percentages of HER2×VEGF bispecific DARPin, anti- HER2 DARPin, and anti-VEGF DARPin by SDS-PAGE analysis before and after treatment with TCEP or BME. HER2xVEGF DARPin After SEC With 1mM TCEP With BMEanti-HER2 DARPin After SEC With 1mM TCEP With BMEanti-VEGF DARPin After SEC With 1mM TCEP With BME- 60 - 12799878v1Attorney Docket No. 2018488-0014

[0174] Monospecific anti-HER2 and anti-VEGF DARPins displayed slightly higher initial dimerization (7.0 % and 7.6 %, respectively), which was similarly reversible upon reducing agent treatment (Table 2). To evaluate scalability, a 10-fold scale-up from a 30 mL culture (1× scale) to a 300 mL culture (10× scale) was performed (FIG. 17A). SDS-PAGE analysis under non-reducing and reducing conditions confirmed that the scale-up did not adversely affect protein purity or integrity (FIG. 17B). Densitometry analysis revealed comparable monomer content and minimal dimer formation (1.9 % vs. 1.6 %), supporting the robustness of the expression and purification processes at larger scales.

[0175] It is worth noting that, to facilitate optional tag removal, our construct was engineered with an N-terminal 6×His-tag followed by a TEV protease cleavage site (FIG. 13B, marked by a black arrowhead). This design allows for efficient, scarless removal of the His-tag when necessary. While the His-tag was retained for the functional studies (such as flow cytometry cell binding and enzyme- linked immunosorbent assay, ELISA) presented herein, the cleavage protocol and purification details are provided in the Methods section to support workflows requiring tag-free protein. Specifically, post- cleavage purification can be efficiently achieved using the similar Ni-NTA affinity chromatography, leveraging the His-tag on TEV protease to separate it and the cleaved tag from the target protein. Furthermore, to ensure suitability for downstream biological applications, all DARPin samples underwent standard endotoxin removal using high-capacity spin columns and verified to contain endotoxin levels below the assay’s detection threshold of 0.5 endotoxin unit (EU) / mL, as detailed in the Methods section.

[0176] To further assess the biochemical stability of the HER2×VEGF bispecific DARPin- Cysteine, a time-course SEC analysis was performed over 21 days at both 4^°C and 25^°C (FIG. 18, Table 3). Storage at 4^°C maintained a predominant monomeric population, with monomer content decreasing slightly from ~100% on Day 0 to 83.9% on Day 21, accompanied by a modest increase in dimer formation from nearly zero to 16.1%. In contrast, samples stored at 25^°C exhibited accelerated dimerization, with monomer content decreasing to 59.5% and dimer increasing to 40.5% by Day 21 (Table 3). Importantly, treatment with 1 mM TCEP at Day 21 effectively restored the DARPin to greater than 99% monomeric species under both storage conditions, confirming that the observed dimerization was mediated by reversible disulfide bond formation. To further validate these findings, - 61 - 12799878v1Attorney Docket No. 2018488-0014 the same samples were analyzed by non-reducing and reducing SDS-PAGE (FIG. 19). Densitometry analysis showed that dimer content at Day 21 was 20.8% for samples stored at 4^°C and 37.6% for samples stored at 25^°C, closely matching the SEC quantification (Table 3). Under reducing conditions, all samples reverted predominantly to monomeric forms, further supporting the disulfide- mediated mechanism of dimerization. Table 3. Quantification of dimer and monomer percentages of HER2×VEGF bispecific DARPin- Cysteine during 21-day storage at 4^°C and 25^°C. Monomer and dimer contents were determined based on SEC analysis in FIG. 18, monitoring protein stability under different storage conditions and confirming the reversible nature of disulfide-mediated dimerization. 4 °C 25 °C

[0177] To complement these stability assessments, thermal denaturation of the HER2×VEGF bispecific DARPin-Cysteine was evaluated by circular dichroism (CD) spectroscopy. CD thermal melting analysis revealed a cooperative unfolding transition with a melting temperature (Tm) of approximately 74.5^°C (FIG. 20), indicating high thermostability and robust α-helical secondary - 62 - 12799878v1Attorney Docket No. 2018488-0014 structure. This thermal profile supports the conformational resilience of the engineered DARPin scaffold and its suitability for downstream biochemical modification and biomedical applications.

[0178] These results collectively demonstrate that the HER2×VEGF bispecific DARPin- Cysteine possesses good storage stability, particularly under refrigerated conditions, and that any dimerization is largely reversible by mild reduction. Overall, the HER2×VEGF bispecific DARPin demonstrated excellent recombinant expression efficiency, high purity after dual-step purification, and strong biochemical stability, providing a robust platform for subsequent bioconjugation and functional evaluation. EXAMPLE 6 - HER2 / VEGF Targeted Binding Assay of DARPins.

[0179] To confirm the retention of dual-targeting capability in the designed HER2×VEGF bispecific DARPin, binding to both HER2 and VEGF using established cell-based and ELISA assays, are evaluated. Monospecific anti-HER2 and anti-VEGF DARPins were used as positive controls, along with commercially available anti-HER2 and anti-VEGF monoclonal antibodies. First, NCI-N87 and BT-474 breast cancer cell lines, which are widely used HER2-positive models, and MDA-MB-231 as a HER2-negative control, are selected. The HER2 expression status of these cell lines was validated by flow cytometry using a commercially available anti-HER2 PE-conjugated monoclonal antibody (PE-mAb). As described herein, NCI-N87 and BT-474 exhibited strong HER2-specific staining, while MDA-MB-231 showed minimal HER2 expression (see Fig. 5 and FIG. 21, panels a–c).

[0180] Then HER2-binding activity of the DARPin constructs are assessed by flow cytometry using an APC-conjugated anti-HisTag monoclonal antibody to detect DARPin binding. The HER2×VEGF bispecific DARPin showed specific binding to HER2-positive NCI-N87 and BT-474 cells, with no detectable binding to HER2-negative MDA-MB-231 cells (FIG. 22). Similarly, the anti- HER2 monospecific DARPin showed selective binding to HER2-positive cells (FIG. 22), whereas the anti-VEGF monospecific DARPin exhibited no binding to any of the tested cell lines (FIG. 22), consistent with the absence of VEGF surface expression on these cell lines.

[0181] In parallel, a VEGF-specific ELISA assay was developed to assess VEGF-binding capability. Wells were pre-coated with recombinant human VEGF165 protein, and DARPin binding was detected using biotinylated anti-HisTag antibody followed by streptavidin-horseradish peroxidase - 63 - 12799878v1Attorney Docket No. 2018488-0014 (HRP) and 3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric detection (FIG. 23). The HER2×VEGF bispecific DARPin demonstrated concentration-dependent binding to VEGF165(ED50=14.4 ng / mL), comparable to the anti-VEGF monospecific DARPin (ED50=5.1 ng / mL), while the anti-HER2 monospecific DARPin showed no clear VEGF binding (ED50 greater than 2000 ng / mL) (FIG. 24). Commercial anti-VEGF mAb-biotin and phosphate-buffered saline (PBS) served as positive and negative controls, respectively, validating the assay specificity. Together, these results confirm that the engineered HER2×VEGF bispecific DARPin retains specific binding to both HER2 and VEGF targets, with comparable performance to monospecific DARPin controls. EXAMPLE 7 - Site-Specific Azide Modification of HER2×VEGF Bispecific DARPin.

[0182] To allow for downstream site-specific conjugation and improve biochemical stability, the free thiol group of the C-terminal cysteine on the HER2×VEGF bispecific DARPin were selectively modified using an azide-PEG3-Maleimide linker. The thiol–maleimide reaction efficiently introduced an azide functionality to the DARPin, generating the HER2×VEGF bispecific DARPin- azide construct (FIG. 25A). Following conjugation, SEC purification was performed to isolate the monomeric DARPin-azide product and remove unreacted linkers or minor byproducts. The final HER2×VEGF bispecific DARPin-azide achieved a monomeric purity greater than 98% as assessed by SEC-high-performance liquid chromatography (HPLC) analysis (FIG. 26), indicating successful modification and excellent product homogeneity.

[0183] The stability of the HER2×VEGF bispecific DARPin-azide was then evaluated over a 23-day period at both 4^°C and 25^°C (FIG. 25B). In contrast to the parent HER2×VEGF bispecific DARPin-Cysteine, which showed progressive dimerization during storage (FIG. 18), the azide- modified DARPin demonstrated remarkable biochemical stability, with minimal dimer formation even under elevated temperature conditions. Quantitative analysis of dimer content at 25^°C revealed a striking difference: while the unmodified DARPin-Cysteine accumulated up to 40.5% dimer content by Day 21 (FIG. 18), the DARPin-azide maintained less than 5% dimer even after 23 days at 25^°C (FIG. 25C). This significant suppression of dimerization is attributed to successful blocking of the reactive C-terminal cysteine via thiol–maleimide chemistry, preventing disulfide-mediated intermolecular crosslinking. Overall, these results confirm that site-specific azide-PEG3-Maleimide modification not - 64 - 12799878v1Attorney Docket No. 2018488-0014 only allows for orthogonal click chemistry conjugation but also dramatically enhances the biochemical stability and homogeneity of the HER2×VEGF bispecific DARPin under storage conditions. EXAMPLE 8 - Proof-of-Concept Bioconjugation of HER2×VEGF Bispecific DARPin for Medical Applications.

[0184] To expand the utility of the engineered HER2×VEGF bispecific DARPin, its compatibility with orthogonal click chemistry-mediated conjugations has been demonstrated. Specifically, the azide-functionalized HER2×VEGF bispecific DARPin (HER2×VEGF DARPin-azide) allowed for efficient strain-promoted azide–alkyne cycloaddition (SPAAC) reactions with various dibenzocyclooctyne (DBCO)-functionalized moieties, including fluorescent dyes, radiometal chelators, drug-linkers, and nanoparticle platforms (FIG. 1, Table 4). This modular conjugation approach offers a versatile platform for biomedical applications, including, but not limited to, optical imaging, positron emission tomography (PET) imaging, targeted radionuclide therapy, and advanced nanodrug delivery strategies. - 65 - 12799878v1Attorney Docket No.2018488-0014 Table 4. Commercially available and custom-designed DBCO-functionalized reagents for click chemistry conjugation with azide- functionalized HER2×VEGF bispecific DARPin. A select list of DBCO-modified molecules, including fluorescent dyes, radiometal chelators, cleavable drug linkers, nanoparticle platforms, and binders for modular DARPin engineering, allowing for a diverse diagnostic and therapeutic applications. C 1 F d 2 R c N 3 d p 4 C dor targeted t erapy - 66 - 12799878v1Attorney Docket No.2018488-0014 O 5 o s b- 67 - 12799878v1Attorney Docket No. 2018488-0014

[0185] The successful site-specific conjugation of HER2×VEGF bispecific DARPin-azide to diverse DBCO-functionalized moieties—including fluorophores, chelators, and a cleavable MMAE drug linker—was validated by both SEC-HPLC and SDS-PAGE analyses. SEC-HPLC profiles (FIG. 27) demonstrated distinct left-shifted elution peaks for all conjugated DARPins compared to the unmodified DARPin-azide and DARPin-Cysteine controls, indicating increased apparent molecular weight consistent with successful covalent coupling. All conjugates show dominant monomeric purity. A minor dimer peak consistently observed across all profiles likely originates from a small fraction (less than 2%) of unconjugated HER2×VEGF DARPin-Cysteine (FIG. 26) and was retained in all bio conjugated samples. Complementary SDS-PAGE analysis under non-reducing and reducing conditions (FIG. 28) further supported the generation of well-defined, high-purity conjugates. Each functionalized variant displayed a clean banding pattern with no evidence of degradation or side- product formation, underscoring the robustness and orthogonality of the click chemistry strategy. FIG. 29 further demonstrates efficient and specific bioconjugation of BP Fluor 647 to the HER2×VEGF bispecific DARPin, as confirmed by co-elution of absorbance peaks at both 280 nm and 651 nm during SEC-HPLC dual-wavelength analysis. These data collectively demonstrate that the C-terminal azide- engineered HER2×VEGF bispecific DARPin serves as a versatile scaffold for generating homogeneous, high-purity conjugates suitable for diverse biomedical applications. EXAMPLE 9 - SPR Assay Development and Validation of Monospecific DARPins.

[0186] To accurately characterize the bispecific binding behavior of HER2×VEGF DARPins and assess the impact of site-specific bioconjugation, a surface plasmon resonance (SPR) assay using monospecific DARPin controls was first established and validated. This step ensured that the immobilized HER2 and VEGF165antigens were functionally active and selectively recognized by their respective DARPins. Recombinant human HER2 or VEGF165proteins were immobilized independently on carboxymethylated dextran sensor chip (CM5) via standard amine coupling, and analytes were injected at concentrations ranging from 0.16 to 160^nM. The binding data were analyzed using a 1:1 Langmuir binding model to extract kinetic parameters (kon, koff) and equilibrium dissociation constants (KD). - 68 - 12799878v1Attorney Docket No. 2018488-0014

[0187] As shown in FIG.30, panels a-h, the anti-HER2 monospecific DARPin exhibited strong, specific binding to the HER2-coated surface (KD= 1.16×10-10M, or 116 pM), with no detectable interaction on VEGF165-coated chips. Conversely, the anti-VEGF DARPin bound exclusively to VEGF165 (KD = 1.23×10-10M, or 123 pM) and showed no response on HER2- immobilized surfaces. These results confirm the specificity of the DARPins and validate that each chip surface was appropriately prepared and functionally selective. This assay validation provides a robust foundation for subsequent kinetic analyses of HER2×VEGF bispecific DARPins. By immobilizing HER2 and VEGF165antigens separately, we can resolve and quantify binding to each target independently. This platform allows for precise assessment of bispecific binding behavior in both unmodified and bioconjugated DARPins and supports direct comparisons of binding affinity and kinetics across a variety of functional modifications. In all subsequent studies, kinetic constants were extracted using the validated 1:1 Langmuir model, ensuring consistent and reproducible analysis of binding interactions. EXAMPLE 10 - Dual Antigen Targeting by Functional HER2×VEGF Bispecific DARPin Bioconjugates.

[0188] To evaluate the dual-targeting capability of HER2×VEGF bispecific DARPins engineered with modular click-reactive sites, we performed SPR assays using the validated CM5 sensor chips with independently immobilized HER2 or VEGF165 antigens (FIG. 31). Following the assay development strategy established earlier, we characterized binding responses of several bioconjugated DARPin variants functionalized for molecular imaging or therapeutic use, including fluorophores, radiometal chelators, and cytotoxic drug-linkers (FIG. 31, panels b–m). Representative kinetic constants are summarized in Table 5.

[0189] The high-affinity binding of unmodified HER2×VEGF DARPin-Cysteine and its site- specifically modified derivative, DARPin-azide were first confirmed. Both constructs demonstrated dual-target engagement with picomolar-to-subnanomolar equilibrium dissociation constants: KD= 77.4 pM for HER2 and 338 pM for VEGF165in the DARPin-Cysteine construct. These values represent potent antigen engagement, in line with reported high-affinity DARPins against HER2 and VEGF. Importantly, the binding affinities of both DARPin constructs compared favorably with those of - 69 - 12799878v1Attorney Docket No. 2018488-0014 commercially available biotinylated monoclonal antibodies: the anti-HER2 antibody (KD= 524 pM) and anti-VEGF antibody (KD= 52 pM) (Table 5). Notably, sensorgrams for these two constructs displayed extremely slow dissociation phases (FIG. 32, panels c–g), a known limitation in SPR analysis of very strong binders like DARPins. These observations reinforce the robustness of the paratope architecture, even after modular cysteine-azide substitution.

[0190] Subsequent SPR analyses of functionally modified HER2×VEGF bispecific DARPins— including conjugates bearing BP Fluor 647 dye, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid), deferoxamine (DFO), and a cleavable MMAE linker—demonstrated preserved binding to both HER2 and VEGF165, with equilibrium dissociation constants (KD) ranging from approximately 1 nM to 3 nM (Table 5). Compared to the parent DARPin-Cysteine, the conjugates retained comparable association rates (kon), while exhibiting modestly faster dissociation rates (koff), accounting for the slight reduction in overall affinity. Despite this shift, all constructs maintained clinically relevant low-nanomolar binding capacity for both targets. These results indicate that site- specific click-based functionalization at the engineered distal cysteine site imposes minimal structural or steric disruption to the antigen-binding interface. This kinetic trend is consistent with prior studies of engineered DARPins, such as PEGylated and dye-labeled variants targeting epithelial cell adhesion molecule (EpCAM), which also showed preserved specificity and only minor changes in functional affinity following bioconjugation. Together, these findings support the robustness of our HER2×VEGF bispecific DARPin scaffold and validate its modular architecture as a versatile platform for targeted imaging and therapeutic applications. - 70 - 12799878v1Attorney Docket No.2018488-0014 Table 5. SPR analysis of HER2 and VEGF binding kinetics of DARPins before and after bioconjugation. HE HE HE HE HE HE ant ant- 71 - 12799878v1Attorney Docket No.2018488-0014 an a- 72 - 12799878v1Attorney Docket No. 2018488-0014

[0191] Building on these target-binding characteristics, the engineered HER2×VEGF bispecific DARPin bioconjugates demonstrate strong translational potential driven by their site-specific conjugation to diverse functional payloads. For example, the HER2×VEGF DARPin-DOTA conjugate (FIG. 31, panels e-g) represents a clinically actionable candidate for targeted radioisotope delivery. Following radiolabeling with therapeutic isotopes such as Lutetium-177 (177Lu, t1 / 2=6.6 days, beta emitter) or Actinium-225 (225Ac, t1 / 2= 9.9 days, alpha emitter), these conjugates can deliver both imaging and therapeutic payloads—offering a tri-functional strategy encompassing HER2 blockade, VEGF sequestration, and radioisotope-based cytotoxicity. This trivalent therapeutic mechanism aligns with emerging paradigms in precision oncology that combine tumor targeting with multimodal payloads to enhance efficacy and reduce systemic toxicity. However, like radio-ligand therapy (RLT), current DARPin-based radiotherapy (Radio-DARPin-Therapy, RDT) strategy still suffers from elevated kidney retention due to renal clearance of small proteins. To address this, emerging nanocarrier systems, offer a promising strategy to reduce off-target uptake and extend systemic circulation, potentially improving therapeutic indices.

[0192] In the case of the monomethyl auristatin E (MMAE)-conjugated bispecific DARPin (FIG. 31, panels k-m), this construct uniquely integrates VEGF neutralization, HER2 antagonism, and site-specific delivery of a cleavable cytotoxic drug linker, allowing for receptor-mediated internalization and lysosomal drug release. Such “multi-mechanistic bioconjugates” may overcome resistance mechanisms associated with single-target ADC and provide greater selectivity through cooperative binding to dual-expressing tumor phenotypes. While effective in site-specific payload delivery, the use of a single cysteine for conjugation limits the drug-to-protein ratio (DAR = 1). This constraint could be addressed by leveraging nanoparticle platform equipped with multiple DBCO moieties, thereby allowing for multivalent DARPin conjugation and higher drug payloads per construct. Such a design could amplify cytotoxic potency while maintaining controlled delivery.

[0193] Beyond the demonstrated conjugation with small-molecule fluorophores, radionuclide chelators, and cleavable cytotoxic drug linkers, the HER2×VEGF bispecific DARPin-azide also offers considerable promise for nanoparticle-based delivery applications. Its compact molecular size (~30 kDa), comparable to single-chain variable fragments (scFvs), makes it well-suited for integration with DBCO-functionalized nanomaterials. For instance, ultrasmall Cornell Prime Dots (C′ dots), which are - 73 - 12799878v1Attorney Docket No. 2018488-0014 currently under clinical investigation for targeted imaging and drug delivery, can be readily conjugated via copper-free click chemistry. Similarly, commercially available DBCO-coated gold nanoparticles (e.g., from Nanopartz) broaden the bioconjugation landscape. While our preliminary studies confirm the feasibility of generating HER2×VEGF bispecific DARPin–gold nanoconjugates, in-depth analysis of surface ligand density, pharmacokinetics, and multivalent binding effects can be addressed in a forthcoming dedicated study, as their complexity exceeds the current scope.

[0194] Altogether, these results establish the HER2×VEGF bispecific DARPin as a versatile and modular scaffold for site-specific conjugation to a wide range of biomedical payloads via orthogonal click chemistry. In addition to small-molecule conjugates, this approach has been extended to protein–protein conjugation using azide- or DBCO-functionalized DARPins, allowing the construction of multi-specific formats not readily achievable through recombinant expression alone. The preserved high-affinity binding to both HER2 and VEGF following conjugation underscores the structural integrity and functional resilience of the DARPin scaffold. The present disclosure specifically incorporates the anti-HER2 DARPin G3 and anti-VEGF DARPin Abicipar pegol — two of the most clinically validated DARPin modules, with G3 originally developed by the Plückthun group. Applying the same modular assembly and site-specific chemistry, other validated or novel monospecific DARPins can be integrated to construct customized bispecific or multispecific agents targeting a broad spectrum of oncogenic pathways. This flexibility provides a powerful and adaptable framework for developing next-generation agents for targeted imaging, therapy, and precision oncology. EXAMPLE 11 - Cloning, Expression, and Purification.

[0195] The gene encoding the bispecific HER2×VEGF DARPin was synthesized based on sequences from prior publications and improved for E. coli expression. The construct was designed with an N-terminal His-tag followed by a TEV protease cleavage site for affinity purification, while a C-terminal GGGSGGGSC linker was incorporated to provide a single cysteine residue for site-specific modification. The gene was cloned into the pET28a(+) vector under the control of a T7 promoter and transformed into E. coli BL21(DE3) cells for recombinant expression. Similar procedures were applied to the expression and purification of the single anti-HER2 and anti-VEGF DARPins. - 74 - 12799878v1Attorney Docket No. 2018488-0014

[0196] Transformed E. coli cultures were grown in Luria–Bertani (LB) medium supplemented with 50 µg / mL kanamycin at 37°C until the optical density at 600 nm (OD₆₀₀) reached 0.6–0.8. Protein expression was induced by adding 0.5 mM IPTG, and the cultures were incubated at 37°C for 3–4 hours. Cells were harvested by centrifugation (4000×g, 15 min, 4°C) and resuspended in lysis buffer (20 mM Sodium Phosphate, pH 7.4, 500 mM NaCl, 10 mM imidazole) supplemented with cOmplete™, Mini, ethylenediaminetetraacetic acid (EDTA)-free Protease Inhibitor Cocktail to prevent proteolysis and lysozyme (0.5 mg / mL) to aid in cell lysis. Lysis was performed by sonication on ice, followed by centrifugation (15,000×g, 45 min, 4°C) to remove cellular debris.

[0197] The clarified supernatant was loaded onto a Ni-NTA affinity column pre-equilibrated with lysis buffer. The column was washed with wash buffer (20 mM Sodium Phosphate, pH 7.4, 500 mM NaCl, 20 mM imidazole) to remove non-specifically bound proteins. The His-tagged DARPin was eluted using elution buffer (20 mM Sodium Phosphate, pH 7.4, 500 mM NaCl, 500 mM imidazole). Eluted fractions were immediately subjected to buffer exchange into 20 mM Sodium Phosphate (pH 7.4) containing 500 mM NaCl using a centrifugal filtration device or dialysis to remove excess imidazole. The protein solution was then passed through a 0.22 µm filter to remove potential aggregates or particulates before further analysis. Protein purity was assessed by SDS-PAGE, and the oligomeric state was analyzed by SEC-HPLC to confirm the presence of monomeric and dimeric species before proceeding with further purification or functional assays. EXAMPLE 12 - Size Exclusion Chromatography (SEC).

[0198] To assess the purity, molecular weight distribution, and aggregation status of the bispecific HER2×VEGF DARPin, as well as the single aHER2 and anti-VEGF DARPins, both analytical and preparative SEC were performed. For analytical SEC, an Agilent 1260 Infinity II LC System was used with a high-resolution SEC column (250 Å, 5 μm, 7.8 mm × 300 mm, molecular weight range 10K–500K) from Waters Technologies Corporation. The column was equilibrated with at least five column volumes of 1× PBS (pH 7.4), and baseline UV absorbance at 280 nm was monitored to ensure stability. Protein samples were filtered prior to injection, and 10–100 µL of sample was loaded at a flow rate of 0.5–1.0 mL / min. Elution was monitored by UV absorbance at 280 nm, and retention times were compared against Bio-Rad Gel Filtration Standards to estimate molecular weight and evaluate the presence of monomeric and dimeric species. Since the bispecific and single - 75 - 12799878v1Attorney Docket No. 2018488-0014 DARPins contain a C-terminal cysteine, dimer formation due to disulfide bonding was anticipated. To assess the reducibility of these dimers, SEC was also performed on samples pretreated with TCEP to reduce disulfide bonds and restore free thiol groups.

[0199] For large-scale SEC purification, a Superdex 200 Increase 10 / 300 GL column was used to separate monomeric and dimeric species of the bispecific and single DARPins. The column was equilibrated with 1× PBS (pH 7.4) with continuous monitoring of the 280 nm baseline to ensure system stability. Typically, 100–900 µL of protein sample was injected at a flow rate of 1.0 mL / min. To maximize protein yield, both monomeric and dimeric fractions were collected. These fractions were analyzed by SDS-PAGE under reducing and non-reducing conditions to confirm the presence of disulfide-linked dimers and assess the effect of TCEP treatment in reverting dimers back to monomers. Collecting both monomeric and dimeric species ensured higher overall DARPin recovery while allowing subsequent reduction steps to prepare monomeric species for downstream applications, such as functional assays and site-specific bioconjugation. (See FIG. 18, FIG. 25B, FIGs. 26-27, FIG. 29, and FIG. 33) EXAMPLE 13 - SDS-PAGE Analysis.

[0200] Purified proteins, including both the single (anti-HER2 and anti-VEGF) and bispecific HER2×VEGF DARPins, were analyzed by SDS-PAGE under reducing and non-reducing conditions to verify molecular weight and assess potential aggregation. Protein samples were mixed with 2× Laemmli buffer, heated at 95°C for 5 minutes, and loaded onto an 8–16% polyacrylamide gel (BioRad, cat#456-1105). Electrophoresis was performed at 120 V for 45 minutes. Gels were stained with Coomassie Brilliant Blue to visualize protein bands. The observed molecular weights were compared to reference values based on sequence-predicted sizes, and the presence of any higher-order aggregates was noted. Gel images were acquired using a Bio-Rad imaging system. Band intensities corresponding to monomeric and dimeric HER2×VEGF bispecific DARPins were quantified using ImageJ software (NIH). The integrated density of each band was measured, and the percentage of monomer and dimer species was calculated by normalizing the individual band intensities to the total lane intensity. - 76 - 12799878v1Attorney Docket No. 2018488-0014 EXAMPLE 14 - Endotoxin Removal and Detection for DARPins.

[0201] To ensure the HER2xVEGF bispecific DARPin, as well as the monospecific anti-HER2 and anti-VEGF DARPins, are free of endotoxins that could interfere with biological assays, DARPin samples should first be prepared and processed using Pierce™ High-Capacity Endotoxin Removal Spin Columns (cat# 88274) equilibrated with endotoxin-free water according to the manufacturer’s instructions. Apply up to 0.5-2 mL of each DARPin sample to the column, incubate at room temperature for 1 hour to allow binding, then centrifuge at 500 × g for 2 minutes to collect the purified sample into a new endotoxin-free tube. Endotoxin levels were then be verified using the Pierce™ Rapid Gel Clot Endotoxin Assay Kit (cat# A43882, 0.5^ EU / mL sensitivity) following the manufacturer’s protocol, by mixing 0.5^mL of sample with the supplied reagent, transferring 0.25^mL to a Positive Control tube, and incubating both tubes at 37^°C for 60 minutes before assessing clot formation. Throughout the procedure, use only endotoxin-free consumables and confirm incubation temperatures with a calibrated thermometer to ensure assay reliability. EXAMPLE 15 - In Vitro Cell Binding Assay.

[0202] To evaluate the binding specificity of the bispecific HER2×VEGF DARPin, HER2- positive BT-474 and NCI-N87 cell lines and HER2-negative MDA-MB-231 control cells were used. Cells were harvested, counted, and resuspended at a concentration of 1 million cells / mL. Cells were then incubated with 10 nM of either the bispecific DARPin or the respective monospecific DARPins (anti-HER2 or anti-VEGF) at 4°C for 30 minutes to allow binding. After incubation, cells were washed and subsequently incubated with 2.5 µL of anti-HisTag APC-conjugated monoclonal antibody for 30 minutes at 4°C to detect DARPin binding. Following antibody incubation, cells were washed again and stained with Zombie NIR™ Fixable Viability Kit (BioLegend, cat# 423105) to exclude non- viable cells. After a final wash, samples were resuspended in PBS (with 2% FBS) and analyzed using a BD FACSymphony™ A1 flow cytometer. Gating was performed to exclude debris and dead cells, ensuring analysis of only viable single-cell populations. EXAMPLE 16 - Enzyme-Linked Immunosorbent Assay (ELISA).

[0203] A quantitative ELISA assay was developed to evaluate the binding of the HER2×VEGF bispecific DARPin to recombinant human VEGF165, with a biotinylated anti-VEGF antibody serving as - 77 - 12799878v1Attorney Docket No. 2018488-0014 a positive control. Additionally, anti-HER2 and anti-VEGF single DARPins were included as control groups to assess binding specificity. High-binding 96-well plates were coated with VEGF165(2.5 µg / mL, 50 µL per well) and incubated overnight at 4°C, followed by blocking with 1% BSA in PBS for 1 hour at room temperature (RT). Serial dilutions of the DARPins (0.5–500 ng / mL, 50 µL per well) and the positive control antibody were prepared and incubated for 1 hour at RT. After washing, bound proteins were detected using biotinylated anti-HisTag monoclonal antibody (1-2 µg / mL, 50 µL per well), followed by streptavidin-HRP (1:20,000 dilution, 100 µL per well) incubation for 30 minutes at RT. The reaction was developed with TMB substrate (100 µL per well, 5-minute incubation), stopped with 50 µL of ELISA stop solution, and absorbance was measured at 450 nm using a microplate reader. Data were analyzed using GraphPad Prism. EXAMPLE 17 - Azide Group Modification and Purification.

[0204] To introduce an azide moiety for site-specific conjugation on the HER2xVEGF bispecific DARPin-Cystine, dimers were first reduced by incubating the DARPin with 1 mM TCEP in PBS (pH 7.4) at room temperature for 30-60 minutes, ensuring free sulfhydryl groups for conjugation. The reduced DARPin was then reacted with Azido-PEG3-Maleimide at a 20:1 molar ratio (Azido- PEG3-Maleimide:DARPin) in PBS (pH 7.4) at room temperature for 2 hours. The modified DARPin- PEG3-azide was purified using SEC on a Superdex 200 column (mobile phase: 1× PBS, pH 7.4), collecting fractions containing the conjugated DARPin as monitored by UV absorbance at 280 nm. EXAMPLE 18 - Surface Plasmon Resonance Binding Assay.

[0205] SPR experiments were performed using a Biacore 8K instrument (Cytiva) to characterize the binding kinetics of bispecific and monospecific DARPin constructs. Recombinant human HER2 (cat# 1129-ER-050) and VEGF165(cat# VEG-HM065) were immobilized on individual flow cells of a CM5 sensor chip via standard amine coupling chemistry. Each protein was immobilized on separate channels to permit simultaneous analysis. The assay was conducted at 25°C using 1× PBS-P+ running buffer (Cytiva). Analytes—bispecific and monospecific DARPin variants—were serially diluted in running buffer to final concentrations ranging from 0.16 to 160 nM. Each analyte injection consisted of a 120-second association phase followed by a 300-900-second dissociation phase at a flow rate of 30 µL / min. Regeneration between cycles was achieved with 30 seconds of glycine- - 78 - 12799878v1Attorney Docket No. 2018488-0014 HCl (pH 2.0) at the same flow rate. The system collected data at 10 Hz. Binding responses were double-referenced by subtracting both the signal from a reference flow cell and the buffer-only injections. The kinetic parameters—association rate constant (ka or kon), dissociation rate constant (kd or koff), and equilibrium dissociation constant (KD)—were calculated by globally fitting the data to a 1:1 Langmuir binding model using Biacore Insight Evaluation Software (v6.0.7.1750). In some cases, sensorgrams with extremely slow dissociation phases were flagged by the software, and interpretation was made accordingly (see Results / Discussion). Monospecific anti-HER2 and anti-VEGF DARPins were included as positive controls to validate binding specificity. EXAMPLE 19 - Circular Dichroism (CD) Thermal Denaturation of HER2xVEGF Bispecific DARPin.

[0206] Thermal denaturation of the HER2xVEGF bispecific DARPin was monitored by circular dichroism (CD) spectroscopy using a Jasco J-1500 CD spectrometer equipped with a PM-539 detector and MPTC-513 temperature control unit. The protein was prepared at 10 µM in PBS (pH 7.4) and measured in a 1 mm pathlength quartz cuvette. CD ellipticity was recorded at 222 nm, a wavelength sensitive to α-helical content in HER2xVEGF bispecific DARPin. The temperature was ramped from 20^°C to 95^°C at a constant rate of 1^°C per minute, with data acquired every ~0.5^°C using 1-second integration time (D.I.T.). Instrument parameters included a bandwidth of 1.00 nm, auto high-tension voltage, and shutter control in auto mode. Target temperature ±0.05^°C was kept for 1 second to ensure thermal equilibrium before each measurement. The melting temperature (Tm) was determined by fitting the unfolding curve to a two-state model with linear baselines. All DARPin samples were confirmed to be monomeric by analytical size-exclusion chromatography prior to CD analysis. EXAMPLE 20 - Azide-DBCO Click Chemistry-Based Bioconjugation of HER2xVEGF Bispecific DARPin.

[0207] Azide modified HER2xVEGF bispecific DARPin was further conjugated to various functional moieties via SPAAC using azide-DBCO click chemistry. For a typical bioconjugation, HER2xVEGF-PEG3-azide was reacted with DBCO-functionalized molecules at a 2:1 molar ratio (DBCO reagent: DARPin). The reaction was conducted in PBS (pH 7.4) at room temperature for 2 - 79 - 12799878v1Attorney Docket No. 2018488-0014 hours with gentle mixing to facilitate efficient conjugation. Bioconjugation was performed with a range of DBCO-functionalized moieties to allow different applications. BP Fluor 647-DBCO (Em=650 nm, Ex=671 nm, BroadPharm, cat# BP-25584) was conjugated for fluorescent labeling and imaging, while Deferoxamine-DBCO (DFO-DBCO, cat# B-773) or DOTA-PEG5-C6-DBCO (BroadPharm, cat# BP-24070) allowed for radiometal chelation (e.g.,89Zr,68Ga,177Lu and225Ac) for PET / SPECT imaging and / or radiotherapy. To develop DARPin-drug conjugates (DDC) for targeted therapy, DBCO-PEG4- Val-Cit-PAB-MMAE (BroadPharm, cat# BP-25659) was conjugated to allow for site-specific delivery of monomethyl auristatin E (MMAE). Following conjugation, bioconjugates were purified using SEC with PBS (pH 7.4) as the mobile phase to remove unreacted components. The purity of the final conjugates was assessed using HPLC. Functional validation was performed using SPR, ensuring that the DARPin retained its target-binding activity post-conjugation. EXAMPLE 21 - Stability Monitoring of HER2xVEGF Bispecific DARPin-Cystine and DARPin- Azide.

[0208] The stability of the HER2xVEGF bispecific DARPin-Cystine was assessed by monitoring dimer formation during storage, attributed to disulfide bond formation via the engineered C-terminal cysteine. DARPin samples were stored at 4^°C and 25^°C and analyzed at predetermined time points (Day 0, 4, 7, 14, and 21). At each time point, 20–50^µg of sample was subjected to SEC using a Superdex 200 Increase 10 / 300 GL column (Cytiva) equilibrated with PBS (pH 7.4) at a flow rate of 1.0^mL / min. Absorbance at 280^nm was monitored, and monomer and dimer species were identified based on retention times, with peak areas integrated for quantitation. To confirm that dimerization was disulfide-mediated, Day 21 samples were treated with 1^mM TCEP (Thermo Fisher Scientific) at room temperature for 1-2 hours or overnight at 4 °C, followed by SEC analysis. Complete reversion of the dimer to monomer after TCEP treatment confirmed the reversible disulfide linkage. Similar stability monitoring procedures were applied to HER2xVEGF bispecific DARPins functionalized with an azide group. Following conjugation, samples were stored and analyzed in parallel at the similar time points and conditions as the unmodified DARPin-Cystine constructs. - 80 - 12799878v1Attorney Docket No. 2018488-0014 EXAMPLE 22 - TEV Protease Cleavage and Ni-NTA Purification.

[0209] To remove the N-terminal 6xHis-tag, the 6xHisTag-TEV-DARPin-PEG3-azide was incubated with TEV Protease (1 unit per 2^µg DARPin) in TEV cleavage buffer (50^mM Tris-HCl, pH 8.0, 0.5^mM EDTA, 1^mM DTT) at 30^°C for 1 hour, following the manufacturer's protocol (NEB, cat# P8112S). The cleaved DARPin (lacking the N-terminal 6xHis-tag but retaining the C-terminal PEG3- azide) was purified by passing the reaction mixture through a Ni-NTA resin / column, collecting the flow-through fraction containing the desired product. The eluted fraction, containing uncleaved protein and His-tag fragments, was discarded. For further purification, SEC might be applied to remove residual contaminants. EXAMPLE 23 – Mechanism of Action Target Cell Recognition

[0210] The HER2-binding domain of the bispecific DARPin selectively binds to HER2- overexpressing tumor cells, ensuring specificity in targeting. (See FIG. 35) T Cell Engagement

[0211] The CD3-binding domain of the DARPin simultaneously binds to the CD3ε chain on the T cell receptor (TCR) complex of T cells, bringing them into close proximity with the tumor cells. (See FIG. 35) Formation of a T cell–target cell interface

[0212] This dual binding facilitates the formation of an immunological synapse between the T cell and the tumor cell, leading to T cell activation. (See FIG. 35) T Cell Activation and Cytotoxicity

[0213] Upon activation, T cells release perforin and granzymes, inducing apoptosis in the tumor cells. (See FIG. 35) - 81 - 12799878v1Attorney Docket No. 2018488-0014 Improved pharmacokinetic profiles via C’ dot conjugation

[0214] Conjugation of bispecific DARPins to C’ dots improves pharmacokinetics by limiting off-target effects, increasing serum half-life and stability, and enhancing tumor penetration / accumulation. (See FIG. 35) EXAMPLE 24 - HER2×VEGF Bispecific DARPin–Radiotherapeutic Conjugate

[0215] FIG. 36 shows design summary, mechanism of action, target indication, and list of clinical needs that are addressed. EXAMPLE 25 - Next-Gen DARPin–C’ dot Immunoconjugates Multi-specific Precision Targeting of DARPin-C’ dots.

[0216] Modular particle-bound DARPins (e.g., HER2×VEGF, CD3×HER2) allow for dual / triple engagement of key tumor / immune pathways, enhance specificity, improve efficacy, and mitigate suppressive activities in the tumor microenvironment. Functionalization of Immune Cells.

[0217] DARPins can replace single-chain variable fragments (scFvs) in various immune cells, such as CAR T cells. They can be genetically engineered into cells to recognize specific tumor antigens. Clinically Validated Nanocarrier.

[0218] C’ dots are safe, renally clearable, exhibit favorable PK, penetrate solid tumors, and serve as a potent adjuvant-therapy. Theranostic constructs are designed to target one or more tumor antigens and engage immune cells.

[0219] Supports multimodal imaging / therapy, redirects T cells towards tumors, modulates immune cell populations (e.g., CD3-engaging constructs), for use in a variety of applications. Improved Pharmacokinetics.

[0220] C’ dot conjugation improves serum half-times and tumor uptake via a “target-or-clear” mechanism, minimizing off-target exposure. - 82 - 12799878v1Attorney Docket No. 2018488-0014 Click-Ready Conjugation.

[0221] Azide-tagged DARPins support efficient click chemistry with C’ dots, allowing site- specific loading of imaging agents, radioisotopes (e.g.,177Lu), or other cytotoxic drugs. Scalable & Stable.

[0222] Bacterial DARPin expression and robust C’ dot synthesis ensure cost-effective, reproducible production of homogeneous, stable conjugates. EXAMPLE 26 - New single-chain fragments (scFvs) developed for C’ dot conjugation

[0223] Amino acid sequences for all the constructs were obtained from publicly available databases. GenScript codon-improved the amino acid sequences and generated plasmids for each scFv using pcDNA3.4 as the backbone for expression in mammalian ExpiCHO cells. The selected peptide sequence, MGWSCIILFLVATATGVHS, was included upstream of the scfv sequences for secretion of the proteins in the ExpiCHO supernatant; a 10xHis-tag was added downstream for Ni-based purification. ExpiCHO cells were transiently transfected with 0.5 μg / mL of plasmid according to the manufacturer’s (ThermoFisher Scientific) protocol. The transfected cells were cultured at 32°C, 125 rpm and 5% CO2for 7 days. The cells were subsequently centrifuged at 3000g for 30 mins to harvest the supernatant, which was filtered through 0.45 μm filter. The filtered supernatant was pre-mixed with binding buffer (25 mM imidazole+20 mM sodium phosphate 0.35 M NaCl buffer, pH 8). Conditioned supernatant was incubated with 2 mL of HisPur Ni-NTA resin (pre-washed 3x with binding buffer) at r.t. for 30 mins. The resins were washed with wash buffer (40 mM imidazole+20 mM sodium phosphate 0.35 M NaCl buffer pH 8). 0.5 mL fractions were obtained using elution buffer (300 mM imidazole+20 mM sodium phosphate 0.35 M NaCl buffer pH 8). Each eluted fractions were checked on NanoDrop to determine protein concentration before performing SDS-PAGE to ascertain size of the protein samples. The average yield of the scFvs was 1 mg / L. Purified scFv products for C’ dot conjugation (anti-VEGF, anti-MUC16, anti-CD3) were generated for targeting multiple cancer cell types and T cells and for creating BiTEs (i.e., anti-MUC16, anti-CD3) and engineered immune cell products (i.e., TCRs). Anti-VEGF was adapted with cysteine (Cys) for C’ dot conjugation. Anti- muc16 was adapted with cysteine (Cys) for C’ dot conjugation. Anti-CD3 was adapted with cysteine (Cys) for C’ dot conjugation. (See FIGs. 37-39) - 83 - 12799878v1Attorney Docket No. 2018488-0014 Computer System and Network Environment

[0224] In another aspect, the invention is directed to a method for machine learning based design of DARPin (designed ankyrin repeat protein) sequences and / or structures, the method comprising, receiving, by a processor of a computing device, one or more target structures for which the DARPin sequence and / or structure is to be designed (e.g., obtain target structure(s) from the Protein Data Bank (PDB) or other database, and / or predict target structure(s) using AlphaFold2 or other predictive software); receive (and / or select), by the processor, a DARPin scaffold (e.g., retrieve a suitable DARPin scaffold from the PDB and / or identify variable positions for sequence design); simulate, by the processor, docking of the scaffold to the target (e.g., using a docking simulation tool such as HADDOCK (High Ambiguity Driven protein-protein DOCKing), RosettaDock, or ClusPro to dock the scaffold to the target, and / or select a plausible docked pose); and design, by the processor, the one or more DARPin sequences and / or structures (e.g., sequences) using the received (and / or selected) DARPin scaffold and at least one of the received one or more target structures (e.g., generate candidate DARPin sequences that optimize variable positions using tools such as Rosetta or ProteinMPNN, score sequences based on predicted binding affinity, use AlphaFold-Multimer to predict complex structures, and select one or more top candidates including the designed DARPin sequence / structure). In certain embodiments, the method comprises the step of experimentally validating the designed DARPin sequence / structure by synthesizing the designed DARPin sequence / structure (and / or synthesizing other top candidates) and testing binding using experimental methods (e.g., lab screening). In another aspect, the invention is directed to a system for machine learning based design of DARPin (designed ankyrin repeat protein) sequences and / or structures, the system comprising a processor of a computing device; and a memory having instructions stored thereon, wherein the instructions, when executed by the processor, cause the processor to perform the method described in this paragraph. Computer System and Network Architecture

[0225] Certain embodiments described herein make use of computer algorithms in the form of software instructions executed by a computer processor. In certain embodiments, the software instructions include a machine learning module, also referred to herein as artificial intelligence software. As used herein, a machine learning module refers to a computer implemented process (e.g., a software function) that implements one or more specific machine learning techniques, e.g., artificial - 84 - 12799878v1Attorney Docket No. 2018488-0014 neural networks (ANNs), e.g., convolutional neural networks (CNNs), e.g., recursive neural networks, e.g., recurrent neural networks such as long short-term memory (LSTM) or Bilateral long short-term memory (Bi-LSTM), random forest, decision trees, transformers, support vector machines, and the like, in order to determine, for a given input, one or more output values.

[0226] In certain embodiments, machine learning modules implementing machine learning techniques are trained, for example using datasets that include categories of data described herein (e.g., CT images, MRI images, PET images, SPECT images). Such training may be used to determine various parameters of machine learning algorithms implemented by a machine learning module, such as weights associated with layers in neural networks. In certain embodiments, once a machine learning module is trained, e.g., to accomplish a specific task, values of determined parameters are fixed and the (e.g., unchanging, static) machine learning module is used to process new data (e.g., different from the training data) and accomplish its trained task without further updates to its parameters (e.g., the machine learning module does not receive feedback and / or updates). In certain embodiments, machine learning modules may receive feedback, e.g., based on user review of accuracy, and such feedback may be used as additional training data, to dynamically update the machine learning module. In certain embodiments, two or more machine learning modules may be combined and implemented as a single module and / or a single software application. In certain embodiments, two or more machine learning modules may also be implemented separately, e.g., as separate software applications. A machine learning module may be software and / or hardware. For example, a machine learning module may be implemented entirely as software, or certain functions of an ANN module may be carried out via specialized hardware (e.g., via an application specific integrated circuit (ASIC)).

[0227] As shown in FIG. 40, an implementation of a network environment 4000 for use in providing systems, methods, and architectures as described herein is shown and described. In brief overview, referring now to FIG. 40, a block diagram of an exemplary cloud computing environment 4000 is shown and described. The cloud computing environment 4000 may include one or more resource providers 4002a, 4002b, 4002c (collectively, 4002). Each resource provider 4002 may include computing resources. In some implementations, computing resources may include any hardware and / or software used to process data. For example, computing resources may include hardware and / or software capable of executing algorithms, computer programs, and / or computer - 85 - 12799878v1Attorney Docket No. 2018488-0014 applications. In some implementations, exemplary computing resources may include application servers and / or databases with storage and retrieval capabilities. Each resource provider 4002 may be connected to any other resource provider 4002 in the cloud computing environment 4000. In some implementations, the resource providers 4002 may be connected over a computer network 4008. Each resource provider 4002 may be connected to one or more computing device 4004a, 4004b, 4004c (collectively, 4004), over the computer network 4008.

[0228] The cloud computing environment 4000 may include a resource manager 4006. The resource manager 4006 may be connected to the resource providers 4002 and the computing devices 4004 over the computer network 4008. In some implementations, the resource manager 4006 may facilitate the provision of computing resources by one or more resource providers 4002 to one or more computing devices 4004. The resource manager 4006 may receive a request for a computing resource from a particular computing device 4004. The resource manager 4006 may identify one or more resource providers 4002 capable of providing the computing resource requested by the computing device 4004. The resource manager 4006 may select a resource provider 4002 to provide the computing resource. The resource manager 4006 may facilitate a connection between the resource provider 4002 and a particular computing device 4004. In some implementations, the resource manager 4006 may establish a connection between a particular resource provider 4002 and a particular computing device 4004. In some implementations, the resource manager 4006 may redirect a particular computing device 4004 to a particular resource provider 4002 with the requested computing resource.

[0229] FIG. 41 shows an example of a computing device 4100 and a mobile computing device 4150 that can be used to implement the techniques described in this disclosure. The computing device 4100 is intended to represent various forms of digital computers, such as laptops, desktops, workstations, personal digital assistants, servers, blade servers, mainframes, and other appropriate computers. The mobile computing device 4150 is intended to represent various forms of mobile devices, such as personal digital assistants, cellular telephones, smart-phones, and other similar computing devices. The components shown here, their connections and relationships, and their functions, are meant to be examples only, and are not meant to be limiting. - 86 - 12799878v1Attorney Docket No. 2018488-0014

[0230] The computing device 4100 includes a processor 4102, a memory 4104, a storage device 4106, a high-speed interface 4108 connecting to the memory 4104 and multiple high-speed expansion ports 4110, and a low-speed interface 4112 connecting to a low-speed expansion port 4114 and the storage device 4106. Each of the processor 4102, the memory 4104, the storage device 4106, the high- speed interface 4108, the high-speed expansion ports 4110, and the low-speed interface 4112, are interconnected using various busses, and may be mounted on a common motherboard or in other manners as appropriate. The processor 4102 can process instructions for execution within the computing device 4100, including instructions stored in the memory 4104 or on the storage device 4106 to display graphical information for a GUI on an external input / output device, such as a display 4116 coupled to the high-speed interface 4108. In other implementations, multiple processors and / or multiple buses may be used, as appropriate, along with multiple memories and types of memory. Also, multiple computing devices may be connected, with each device providing portions of the necessary operations (e.g., as a server bank, a group of blade servers, or a multi-processor system). Thus, as the term is used herein, where a plurality of functions are described as being performed by “a processor”, this encompasses embodiments wherein the plurality of functions are performed by any number of processors (one or more) of any number of computing devices (one or more). Furthermore, where a function is described as being performed by “a processor”, this encompasses embodiments wherein the function is performed by any number of processors (one or more) of any number of computing devices (one or more) (e.g., in a distributed computing system).

[0231] The memory 4104 stores information within the computing device 4100. In some implementations, the memory 4104 is a volatile memory unit or units. In some implementations, the memory 4104 is a non-volatile memory unit or units. The memory 4104 may also be another form of computer-readable medium, such as a magnetic or optical disk.

[0232] The storage device 4106 is capable of providing mass storage for the computing device 4100. In some implementations, the storage device 4106 may be or contain a computer-readable medium, such as a hard disk device, an optical disk device, a flash memory or other similar solid state memory device, or an array of devices, including devices in a storage area network or other configurations. Instructions can be stored in an information carrier. The instructions, when executed by one or more processing devices (for example, processor 4102), perform one or more methods, such - 87 - 12799878v1Attorney Docket No. 2018488-0014 as those described above. The instructions can also be stored by one or more storage devices such as computer- or machine-readable mediums (for example, the memory 4104, the storage device 4106, or memory on the processor 4102).

[0233] The high-speed interface 4108 manages bandwidth-intensive operations for the computing device 4100, while the low-speed interface 4112 manages lower bandwidth-intensive operations. Such allocation of functions is an example only. In some implementations, the high-speed interface 4108 is coupled to the memory 4104, the display 4116 (e.g., through a graphics processor or accelerator), and to the high-speed expansion ports 4110, which may accept various expansion cards (not shown). In the implementation, the low-speed interface 4112 is coupled to the storage device 4106 and the low-speed expansion port 4114. The low-speed expansion port 4114, which may include various communication ports (e.g., USB, Bluetooth®, Ethernet, wireless Ethernet) may be coupled to one or more input / output devices, such as a keyboard, a pointing device, a scanner, or a networking device such as a switch or router, e.g., through a network adapter.

[0234] The computing device 4100 may be implemented in a number of different forms, as shown in the FIG. 41. For example, it may be implemented as a standard server 4120, or multiple times in a group of such servers. In addition, it may be implemented in a personal computer such as a laptop computer 4122. It may also be implemented as part of a rack server system 4124. Alternatively, components from the computing device 4100 may be combined with other components in a mobile device (not shown), such as a mobile computing device 4150. Each of such devices may contain one or more of the computing device 4100 and the mobile computing device 4150, and an entire system may be made up of multiple computing devices communicating with each other.

[0235] The mobile computing device 4150 includes a processor 4152, a memory 4164, an input / output device such as a display 4154, a communication interface 4166, and a transceiver 4168, among other components. The mobile computing device 4150 may also be provided with a storage device, such as a micro-drive or other device, to provide additional storage. Each of the processor 4152, the memory 4164, the display 4154, the communication interface 4166, and the transceiver 4168, are interconnected using various buses, and several of the components may be mounted on a common motherboard or in other manners as appropriate. - 88 - 12799878v1Attorney Docket No. 2018488-0014

[0236] The processor 4152 can execute instructions within the mobile computing device 4150, including instructions stored in the memory 4164. The processor 4152 may be implemented as a chipset of chips that include separate and multiple analog and digital processors. The processor 4152 may provide, for example, for coordination of the other components of the mobile computing device 4150, such as control of user interfaces, applications run by the mobile computing device 4150, and wireless communication by the mobile computing device 4150.

[0237] The processor 4152 may communicate with a user through a control interface 4158 and a display interface 4156 coupled to the display 4154. The display 4154 may be, for example, a TFT (Thin-Film-Transistor Liquid Crystal Display) display or an OLED (Organic Light Emitting Diode) display, or other appropriate display technology. The display interface 4156 may comprise appropriate circuitry for driving the display 4154 to present graphical and other information to a user. The control interface 4158 may receive commands from a user and convert them for submission to the processor 4152. In addition, an external interface 4162 may provide communication with the processor 4152, so as to allow for near area communication of the mobile computing device 4150 with other devices. The external interface 4162 may provide, for example, for wired communication in some implementations, or for wireless communication in other implementations, and multiple interfaces may also be used.

[0238] The memory 4164 stores information within the mobile computing device 4150. The memory 4164 can be implemented as one or more of a computer-readable medium or media, a volatile memory unit or units, or a non-volatile memory unit or units. An expansion memory 4174 may also be provided and connected to the mobile computing device 4150 through an expansion interface 4172, which may include, for example, a SIMM (Single In Line Memory Module) card interface. The expansion memory 4174 may provide extra storage space for the mobile computing device 4150, or may also store applications or other information for the mobile computing device 4150. Specifically, the expansion memory 4174 may include instructions to carry out or supplement the processes described above, and may include secure information also. Thus, for example, the expansion memory 4174 may be provide as a security module for the mobile computing device 4150, and may be programmed with instructions that permit secure use of the mobile computing device 4150. In addition, secure applications may be provided via the SIMM cards, along with additional information, such as placing identifying information on the SIMM card in a non-hackable manner. - 89 - 12799878v1Attorney Docket No. 2018488-0014

[0239] The memory may include, for example, flash memory and / or NVRAM memory (non- volatile random access memory), as discussed below. In some implementations, instructions are stored in an information carrier. The instructions, when executed by one or more processing devices (for example, processor 4152), perform one or more methods, such as those described above. The instructions can also be stored by one or more storage devices, such as one or more computer- or machine-readable mediums (for example, the memory 4164, the expansion memory 4174, or memory on the processor 4152). In some implementations, the instructions can be received in a propagated signal, for example, over the transceiver 4168 or the external interface 4162.

[0240] The mobile computing device 4150 may communicate wirelessly through the communication interface 4166, which may include digital signal processing circuitry where necessary. The communication interface 4166 may provide for communications under various modes or protocols, such as GSM voice calls (Global System for Mobile communications), SMS (Short Message Service), EMS (Enhanced Messaging Service), or MMS messaging (Multimedia Messaging Service), CDMA (code division multiple access), TDMA (time division multiple access), PDC (Personal Digital Cellular), WCDMA (Wideband Code Division Multiple Access), CDMA2000, or GPRS (General Packet Radio Service), among others. Such communication may occur, for example, through the transceiver 4168 using a radio-frequency. In addition, short-range communication may occur, such as using a Bluetooth®, Wi-Fi™, or other such transceiver (not shown). In addition, a GPS (Global Positioning System) receiver module 4170 may provide additional navigation- and location-related wireless data to the mobile computing device 4150, which may be used as appropriate by applications running on the mobile computing device 4150.

[0241] The mobile computing device 4150 may also communicate audibly using an audio codec 4160, which may receive spoken information from a user and convert it to usable digital information. The audio codec 4160 may likewise generate audible sound for a user, such as through a speaker, e.g., in a handset of the mobile computing device 4150. Such sound may include sound from voice telephone calls, may include recorded sound (e.g., voice messages, music files, etc.) and may also include sound generated by applications operating on the mobile computing device 4150. - 90 - 12799878v1Attorney Docket No. 2018488-0014

[0242] The mobile computing device 4150 may be implemented in a number of different forms, as shown in FIG. 41. For example, it may be implemented as a cellular telephone 4180. It may also be implemented as part of a smart-phone 4182, personal digital assistant, or other similar mobile device.

[0243] Various implementations of the systems and techniques described here can be realized in digital electronic circuitry, integrated circuitry, specially designed ASICs (application specific integrated circuits), computer hardware, firmware, software, and / or combinations thereof. These various implementations can include implementation in one or more computer programs that are executable and / or interpretable on a programmable system including at least one programmable processor, which may be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device.

[0244] These computer programs (also known as programs, software, software applications or code) include machine instructions for a programmable processor, and can be implemented in a high- level procedural and / or object-oriented programming language, and / or in assembly / machine language. As used herein, the terms machine-readable medium and computer-readable medium refer to any computer program product, apparatus and / or device (e.g., magnetic discs, optical disks, memory, Programmable Logic Devices (PLDs)) used to provide machine instructions and / or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term machine-readable signal refers to any signal used to provide machine instructions and / or data to a programmable processor.

[0245] To provide for interaction with a user, the systems and techniques described here can be implemented on a computer having a display device (e.g., a LCD (liquid crystal display) monitor) for displaying information to the user and a keyboard and a pointing device (e.g., a mouse or a trackball) by which the user can provide input to the computer. Other kinds of devices can be used to provide for interaction with a user as well; for example, feedback provided to the user can be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback); and input from the user can be received in any form, including acoustic, speech, or tactile input. - 91 - 12799878v1Attorney Docket No. 2018488-0014

[0246] The systems and techniques described here can be implemented in a computing system that includes a back end component (e.g., as a data server), or that includes a middleware component (e.g., an application server), or that includes a front end component (e.g., a client computer having a graphical user interface or a Web browser through which a user can interact with an implementation of the systems and techniques described here), or any combination of such back end, middleware, or front end components. The components of the system can be interconnected by any form or medium of digital data communication (e.g., a communication network). Examples of communication networks include a local area network (LAN), a wide area network (WAN), and the Internet.

[0247] The computing system can include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

[0248] In some implementations, various modules described herein can be separated, combined or incorporated into single or combined modules. Modules depicted in FIG. 41 are not intended to limit the systems described herein to the software architectures shown therein.

[0249] Elements of different implementations described herein may be combined to form other implementations not specifically set forth above. Elements may be left out of the processes, computer programs, databases, etc. described herein without adversely affecting their operation. In addition, the logic flows depicted in FIG. 41 do not require the particular order shown, or sequential order, to achieve desirable results. Various separate elements may be combined into one or more individual elements to perform the functions described herein.

[0250] Throughout the description, where apparatus and systems are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are apparatus, and systems of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps. - 92 - 12799878v1Attorney Docket No. 2018488-0014

[0251] It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously. - 93 - 12799878v1Attorney Docket No. 2018488-0014 SEQUENCES Anti-HER2 monospecific DARPin (SEQ ID NO. 1) MGHHHHHHENLYFQGSDLGKKLLEAARAGQDDEVRILMANGADVNAKDEYGLTPLYLATAHGHLEIVEV LLKNGADVNAVDAIGFTPLHLAAFIGHLEIAEVLLKHGADVNAQDKFGKTAFDISIGNGNEDLAEILQK LNGGGSGGGSC Anti-VGEF monospecific DARPin (SEQ ID NO. 2) MGHHHHHHENLYFQGSDLDKKLLEAARAGQDDEVRILMANGADVNARDSTGWTPLHLAAPWGHPEIVEV LLKNGADVNAADFQGWTPLHLAAAVGHLEIVEVLLKYGADVNAQDKFGKTAFDISIDNGNEDLAEILQK AAGGGSGGGSC HER2xVEGF bispecific DARPin (SEQ ID NO. 3) MGHHHHHHENLYFQGSDLGKKLLEAARAGQDDEVRILMANGADVNAKDEYGLTPLYLATAHGHLEIVEV LLKNGADVNAVDAIGFTPLHLAAFIGHLEIAEVLLKHGADVNAQDKFGKTAFDISIGNGNEDLAEILQK LNGGGGSGGGGSGGGGSGSDLDKKLLEAARAGQDDEVRILMANGADVNARDSTGWTPLHLAAPWGHPEI VEVLLKNGADVNAADFQGWTPLHLAAAVGHLEIVEVLLKYGADVNAQDKFGKTAFDISIDNGNEDLAEI LQKAAGGGSGGGSC - 94 - 12799878v1

Claims

1. Attorney Docket No. 2018488-0014 CLAIMS We claim:

1. A Designed Ankyrin Repeat Protein (DARPin) nanoparticle conjugate comprising: a nanoparticle; and one or more DARPins conjugated to the nanoparticle, wherein the nanoparticle has a diameter no greater than 20 nanometers.

2. The nanoparticle conjugate of claim 1, wherein the one or more DARPins comprises one or more DARPin binders.

3. The nanoparticle conjugate of claim 2, wherein the one or more DARPin binders comprises a HER2-binding domain.

4. The nanoparticle conjugate of claim 2 or 3, wherein the one or more DARPin binders comprises a CD3-binding domain.

5. The nanoparticle conjugate of any one of claims 2-4, wherein the one or more DARPin binders comprises an EGFR-binding domain.

6. The nanoparticle conjugate of any one of claims 2-5, wherein the one or more DARPin binders comprises an a4-1BB-binding domain.

7. The nanoparticle conjugate of any one of claims 2-6, wherein the one or more DARPin binders comprises an EpCam-binding domain.

8. The nanoparticle conjugate of any one of claims 2-7, wherein the one or more DARPin binders comprises a VEGF binding domain. - 95 - 12799878v1 Attorney Docket No. 2018488-0014 9. The nanoparticle conjugate of any one of claims 2-8, wherein the one or more DARPin binders comprises a HER2-binding domain and a CD3-binding domain.

10. The nanoparticle conjugate of any one of claims 2-9, wherein the one or more DARPin binders comprises a HER2-binding domain and a VEGF-binding domain.

11. The nanoparticle conjugate of any one of claims 2-9, wherein the one or more DARPin binders comprises a HER2-binding domain, an EGFR-binding domain, and a VEGF-binding domain.

12. The nanoparticle conjugate of any one of claims 2-5, wherein the one or more DARPin binders comprises a CD3-binding domain, an EGFR-binding domain, and a HER2-binding domain.

13. The nanoparticle conjugate of any one of claims 2-12, wherein the nanoparticle conjugate penetrates the blood brain barrier (BBB).

14. The nanoparticle conjugate of any one of the preceding claims, wherein the one or more DARPins is covalently or non-covalently bonded to the nanoparticle via a linker or covalently or non- covalently bonded directly to the nanoparticle, or associated with the nanoparticle or a moiety surrounding the nanoparticle.

15. An anti-angiogenic and / or immunomodulatory nanoparticle conjugate (“AAINC”) comprising: a nanoparticle; and one or more antibody fragments conjugated to the nanoparticle, wherein the one or more antibody fragments targets an angiogenic protein, wherein the nanoparticle has a diameter no greater than 20 nanometers.

16. The nanoparticle conjugate of claim 15, wherein the angiogenic protein comprises a VEGF protein. - 96 - 12799878v1 Attorney Docket No. 2018488-0014 17. The nanoparticle conjugate of claim 15 or 16, wherein the one or more antibody fragments comprises anti-VEGF.

18. The nanoparticle conjugate of any one of claims 15-17, wherein the one or more antibody fragments is a member selected from the set consisting of a recombinant antibody fragment (Fabs), a single chain variable fragment (scFv), and a single domain antibody (sdAb) fragment.

19. The nanoparticle conjugate of any one of claims 15-18, wherein the one or more antibody fragments is a single chain variable fragment (scFv).

20. The nanoparticle conjugate of any one of claims 15-19, wherein the one or more antibody fragments is covalently or non-covalently bonded to the nanoparticle via a linker or covalently or non- covalently bonded directly to the nanoparticle, or associated with the nanoparticle or a moiety surrounding the nanoparticle.

21. The nanoparticle conjugate of any one of claims 15-20, wherein the one or more antibody fragments comprises from 1 to 20 antibody fragments conjugated to the nanoparticle.

22. The nanoparticle conjugate of any one of claims 15-21, wherein the one or more antibody fragments comprises a molecular range no greater than 50 kDa.

23. The nanoparticle conjugate of any one of the preceding claims, wherein the average nanoparticle diameter is from 1 to 20 nm. - 97 - 12799878v1 Attorney Docket No. 2018488-0014 24. The nanoparticle conjugate of any one of the preceding claims, wherein the nanoparticle is coated with an organic polymer.

25. The nanoparticle conjugate of any one of the preceding claims, wherein the nanoparticle conjugate comprises a radiolabel.

26. The nanoparticle conjugate of any one of the preceding claims, wherein the nanoparticle conjugate comprises a first chelator.

27. The nanoparticle conjugate of any one of the preceding claims, wherein the nanoparticle comprises silica.

28. The nanoparticle conjugate of any one of the preceding claims, wherein the nanoparticle comprises a silica core.

29. The nanoparticle conjugate of any one of the preceding claims, wherein the nanoparticle comprises a silica-based core and a silica shell surrounding at least a portion of the core.

30. The nanoparticle conjugate of any one of the preceding claims, wherein the nanoparticle comprises a fluorescent compound within the core.

31. The nanoparticle conjugate of any one of the preceding claims, wherein the nanoparticle comprises a silica composition such that ferroptosis is not induced.

32. The nanoparticle conjugate of any one of the preceding claims, wherein the nanoparticles have a silica composition such that ferroptosis, other cell death processes, and apoptosis may be induced. - 98 - 12799878v1 Attorney Docket No. 2018488-0014 33. The nanoparticle conjugate of any one of the preceding claims, further comprising a therapeutic agent.

34. The nanoparticle conjugate of claim 33, wherein the therapeutic agent is associated to the nanoparticle.

35. The nanoparticle conjugate of any one of claims 1-14 or 34, wherein the therapeutic agent is associated with the one or more DARPins or with the one or more DARPins and the nanoparticle.

36. The nanoparticle conjugate of any one of claims 1-14 or 34, wherein the therapeutic agent is associated with the one or more antibody fragments or with the one or more antibody fragments and the nanoparticle.

37. The nanoparticle conjugate of any one of the preceding claims, wherein the nanoparticle further comprises a targeting ligand.

38. A method of treating a disease or condition and / or mitigating the onset and / or progression of a disease for a subject, the method comprising administering to the subject a pharmaceutical composition comprising the nanoparticle conjugate of any one of claims 1 to 37.

39. The method of claim 38, comprising administering a therapeutic radioisotope.

40. The method of claim 38 or 39, comprising administering immunotherapy. - 99 - 12799878v1 Attorney Docket No. 2018488-0014 41. The method of claim 40, wherein the immunotherapy comprises administering to a subject a pharmaceutical composition comprising the nanoparticle conjugate of any one of claims 15 to 37.

42. The method of claim 40 or 41, wherein the immunotherapy comprises administering an engineered immunotherapy comprising: an engineered immune cell.

43. The method of claim 42, wherein the engineered immune cell comprises (a) a chimeric antigen receptor (CAR) and (b) a bispecific immune cell engager.

44. The method of claim 42 or 43, wherein the engineered immune cell comprises a CAR T cell comprising one or more targeting ligands.

45. The method of any one of claims 38-44, wherein the pharmaceutical composition further comprises a carrier.

46. The method of any one of claims 38-45, wherein the subject has a disease or condition.

47. The method of any one of claims 38-46, wherein the subject has received an anti-inflammatory therapy, chemotherapy, radiotherapy, immunotherapy, or engineered cellular therapy.

48. A method of treating a disease or condition and / or mitigating the onset and / or progression of a disease, the method comprising: administering to a subject a first pharmaceutical composition comprising the nanoparticle conjugate of any one of claims 1 to 37, wherein the subject also receives a second pharmaceutical composition. - 100 - 12799878v1 Attorney Docket No. 2018488-0014 49. The method of claim 48, wherein the second composition comprises an anti-angiogenic agent, radiotherapeutic agent, a chemotherapeutic agent, an immunotherapeutic agent, or engineered cells.

50. The method of claim 48 or 49, wherein the first pharmaceutical composition is being used to enhance activity of the second pharmaceutical composition.

51. A method of treating a disease or condition and / or mitigating the onset and / or progression of a disease, the method comprising: administering to a subject a first pharmaceutical composition comprising the nanoparticle conjugate of any one of claims 1 to 37 and a second pharmaceutical composition.

52. The method of claim 51, wherein the second composition comprises an anti-angiogenic agent, a radiotherapeutic agent, a chemotherapeutic agent, an immunotherapeutic agent, or engineered cells.

53. The method of claim 51 or 52, wherein the first pharmaceutical composition is being used to enhance activity of the second pharmaceutical composition.

54. A method of treating a disease or condition and / or mitigating the onset and / or progression of a disease, the method comprising: administering to a subject a first pharmaceutical composition comprising an anti-angiogenic agent, a radiotherapeutic agent, a chemotherapeutic agent, an immunotherapeutic agent, or engineered cells, wherein the subject also receives a second pharmaceutical composition comprising the nanoparticle conjugate of any one of claims 1 to 37. - 101 - 12799878v1 Attorney Docket No. 2018488-0014 55. The method of claim 44, wherein the second pharmaceutical composition is being used to enhance activity of the first pharmaceutical composition.

56. A method of in vivo imaging, the method comprising: administering to a subject a composition comprising the nanoparticle conjugate of any one of claims 1 to 37, wherein the nanoparticle conjugate comprises an imaging agent; and detecting the imaging agent.

57. A method of manufacturing the nanoparticle conjugate of any one of claims 1 to 37, the method comprising: contacting a nanoparticle-Dibenzocyclooctyne (DBCO) with an azide-antibody fragment, thereby producing the nanoparticle conjugate.

58. A composition comprising one or more agents selected from (1) a nanoparticle conjugate of any one of claims 1 to 37, (2) a radiotherapeutic agent, (3) a second anti-angiogenic agent, (4) an immunotherapeutic agent, or (5) a combination thereof in a unit dosage effective to treat a disease or condition in a subject receiving therapy with the agent as part of a combination of (1) to (4).

59. A Designed Ankyrin Repeat Protein (DARPin) nanoparticle conjugate comprising: a nanoparticle; and one or more DARPins, wherein the nanoparticle has a diameter no greater than 20 nanometers for use in therapy.

60. A Designed Ankyrin Repeat Protein (DARPin) nanoparticle conjugate comprising: a nanoparticle; and one or more DARPins, - 102 - 12799878v1 Attorney Docket No. 2018488-0014 wherein the nanoparticle has a diameter no greater than 20 nanometers for use in a method of treating a disease or condition and / or mitigating the onset and / or progression of a disease in a subject, wherein the treating comprises: delivering the nanoparticle conjugate to the subject.

61. A Designed Ankyrin Repeat Protein (DARPin) nanoparticle conjugate comprising: a nanoparticle; and one or more DARPins, wherein the nanoparticle has a diameter no greater than 20 nanometers for use in a method of in vivo diagnosis of a disease or condition in a subject, wherein the in vivo diagnosis comprises: delivering the nanoparticle conjugate to the subject; and detecting the imaging agent.

62. An anti-angiogenic and / or immunomodulatory nanoparticle conjugate (“AAINC”) comprising: a nanoparticle; and one or more antibody fragments conjugated to the nanoparticle, wherein the nanoparticle has a diameter no greater than 20 nanometers for use in therapy.

63. An anti-angiogenic and / or immunomodulatory nanoparticle conjugate (“AAINC”) comprising: a nanoparticle; and one or more antibody fragments conjugated to the nanoparticle, wherein the nanoparticle has a diameter no greater than 20 nanometers for use in a method of treating a disease or condition and / or mitigating the onset and / or progression of a disease in a subject, wherein the treating comprises: delivering the AAINC to the subject. - 103 - 12799878v1 Attorney Docket No. 2018488-0014 64. An anti-angiogenic and / or immunomodulatory nanoparticle conjugate (“AAINC”) comprising: a nanoparticle; and one or more antibody fragments conjugated to the nanoparticle, wherein the nanoparticle has a diameter no greater than 20 nanometers for use in a method of in vivo diagnosis of a disease or condition in a subject, wherein the in vivo diagnosis comprises: delivering the AAINC to the subject; and detecting the imaging agent.

65. A treatment comprising: (a) a therapeutically effective amount of the nanoparticle conjugate of any one of claims 1 to 37 for use in combination with one or more of the following: (1) an anti-angiogenic agent, (2) a radiotherapeutic agent, (3) a chemotherapeutic agent, (4) an immunotherapeutic agent, or (5) engineered cells, or (b) a therapeutically effective amount of one or more of the following: (1) an anti-angiogenic agent, (2) a radiotherapeutic agent, (3) a chemotherapeutic agent, (4) an immunotherapeutic agent, or (5) engineered cells for use in combination with the nanoparticle conjugate of any one of claims 1 to 37, for use in a method of treating a disease or condition and / or preventing disease or condition occurrence or recurrence in a subject and / or mitigating the onset and / or progression of a disease comprising: an engineered immune cell comprising (a) a chimeric antigen receptor (CAR) and (b) a bispecific immune cell engager. - 104 - 12799878v1

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